Process for the production of fine chemicals

ABSTRACT

The present invention relates to a process for the production of the fine chemical in a microorgansm, a plant cell, a plant, a plant tissue or in one or more parts thereof. The invention furthermore relates to nucleic acid molecules, polypeptides, nucleic acid constructs, vectors, antisense molecules, antibodies, host cells, plant tissue, propagtion material, harvested material, plants, microorganisms as well as agricultural compositions and to their use.

The present invention relates to a process for the production of thefine chemical in a microorgansm, a plant cell, a plant, a plant tissueor in one or more parts thereof. The invention furthermore relates tonucleic acid molecules, polypeptides, nucleic acid constructs, vectors,antisense molecules, antibodies, host cells, plant tissue, propagtionmaterial, harvested material, plants, microorganisms as well asagricultural compositions and to their use.

Certain products and by-products of naturally-occurring metabolicprocesses in cells have utility in a wide array of industries,including, but not limited to, the food, feed, cosmetics, andpharmaceutical industries and agriculture. These molecules, collectivelytermed ‘fine chemicals’ include molecules such as vitamins for examplevitamin A, D, E, K, B₁, B₂, B₆, B₁₂, C, pantothenic acid, biotin orfolic acid; substances with vitamin-like character for example vitaminF, lipoic acid, ubiquinones, choline, myoinsositiol, vitamin U(S-methylmethionine), flavours for example vanillin, coumarin,isoeugenol, eugenol, (R)-carvone, (S)-carvone, menthol, jasmone orfarnesol; nutraceuticals for example phytosterols, flavonoids,anthocyanidins, isoflavons or isoprenoids; detergents; fatty acids suchas saturated fatty acids, mono unsaturated fatty acids (singular MUFA,plural MUFAS), poly unsaturated fatty acids (singular PUFA, pluralPUFAS), waxes or lipids containing said fatty acids; carbohydrates forexample cellulose, starch, dextrin, pectin, xanthangum, carrageenan oralginate; sugars for example monosaccharides such as glucose, fructose,manose, sorbose, ribose, ribulose, xylose, xylulose or galactose,disaccharides such as lactose, sucrose, saccharose, maltose, isomaltoseor cellobiose, trisaccharides such as raffinose or maltotriose;carboxylic acids for example citric acid, α-ketoglutaric acid, ferulicacid, sinapic acid or lactic acid; carotinoids for example α-carotene,β-carotene, zeaxanthine, lutein, astaxanthine, lycopene, phyotoene orphytofluene, amino acids for example lysine, threonine, methionine,tryptophane, phenylalanine or tyrosine, cofactors for example heme orquinines, enzymes for example lipases, esterases, proteases, amylases,glucosidases etc. and other compounds [as described e.g. in Kuninaka, A.(1996) Nucleotides and related compounds, p. 561-612, in Biotechnologyvol. 6, Rehm et al., eds. VCH: Weinheim, in Industial Microbiology andBiotechnology, Demain et al., second edition, ASM Press Washington, D.C.1999, in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27,Vitamins, p. 443-613 (1996) VCH: Weinheim and Ong, A. S., Niki, E. &Packer, L. (1995) Nutrition, Lipids, Health, and Disease Proceedings ofthe UNESCO/Confederation of Scientific and Technological Associations inMalaysia, and the Society for Free Radical Research, Asia, held Sep.1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, and allother chemicals described in Gutcho (1983) Chemicals by Fermentation,Noyes Data Corporation, ISBN: 0818805086 and references containedtherein]. Carotinoids are added for example to soft drinks, margarinesor to animal feed for example to colour egg yolk or the flesh of fish.In the food industry polycarbohydrates are widely used as thickener.Polyunsaturated fatty acids are added for example to infant formulas tocreate a higher nutrition value of such formulas. PUFAs have for examplea positive influence on the cholesterol level of the blood in humans andtherefore are useful in the protection of heart diseases. Fine chemicalsfor example PUFAS can be isolated from animal sources such as forexample fish or produced with microorganisms through the large-scaleculture of microorganisms developed to produce and accumulate or secretelarge quantities of one or more desired molecules.

In large scale fine chemicals are produced with microorganism in thefermentation industry, which is responsible for the manufacturing of atleast five major ingredient categories: antibiotics, organic acids,amino acids, enzymes, vitamins and other related products. There areproduction facilities in all important areas of the world especially inEurope, the US and Asia. Companies continuously try to optimize theproduction processes, the organisms and thereby increasing theefficiency but, as in the case of amino acids and organic acids, withalready high conversion rates based on feeded carbon source, thelimitations of such work become evident. All fermentation processesdepend on the efficient utilization of carbohydrates, supplied mainly inthe form of oils, glucose or molasses. It is therefore the availabilityand pricing of these raw materials that influence the competitiveness offermentation products versus production for example in plants. Aminoacids, organic acids and vitamins are offered at very low prices. Forsuch products the question is whether it is still economical to continuefermentation production in future. And that is frequently a question ofcomparing the availability and pricing of carbohydrates with the futuremarkets.

Particularly useful organisms for the production of fine chemicals aremicroorganisms such as the algae, fungi, bacteria or plants. Throughstrain selection, a number of mutant strains of microorganisms have beendeveloped which produce an array of desirable compounds includingvitamins, amino acids, PUFAs etc. However, selection of strains improvedfor the production of a particular molecule is a time-consuming anddifficult process.

Alternatively the production of fine chemicals can be most convenientlyperformed via the large scale production of plants developed to producefor example carotinoids, carbohydrates or PUFAS. For example for theproduction of carotinoids plants such as marigold are used. Particularlywell-suited plants for this purpose are sugar producing plants such assugar beet or sugar cane or oilseed plants containing high amounts oflipid compounds such as rapeseed, canola, linseed, soybean, sunflower,borage and evening primrose. But also other crop plants containingsugars, oils or lipids and fatty acids are well suited as mentioned inthe detailed description of this invention. Through conventionalbreeding, a number of mutant plants have been developed which produce anarray of desirable lipids and fatty acids, cofactors and enzymes.However, selection of new plant cultivars improved for the production ofa particular molecule is a time-consuming and difficult process or evenimpossible if the compound does not naturally occur in the respectiveplant as for example in the case of C₂₀ and higher C-carbon chainpolyunsaturated fatty acids.

Carbohydrates are an important dietary nutrient, which is mostly used tosupply energy to the body, as well as, a carbon source for synthesis ofother compounds such as fats or proteins. Furthermore mono- anddisaccharides are widely used in the food and feed industry assweetener. Saccharides have varying degrees of sweetness on a relativescale. Fructose is the sweetest. For example in the United States 22million tons of sugar and other sweeteners were consumed in 1999. Saidnatural sweetener consumption includes the consumption of sugar, cornsweeteners such as high fructose corn syrup as main product and otherssuch as honey or maple syrup. All natural sugar based sweeteners have amarket shar of around 36 to 40 percent.

Whether unsaturated or saturated fatty acids are preferred in the foodand feed industry depends on the intended purpose; thus, for example,lipids with unsaturated fatty acids, specifically polyunsaturated fattyacids, are preferred in human nutrition since they have a positiveeffect on the cholesterol level in the blood and thus on the possibilityof heart disease. They are used in a variety of dietetic foodstuffs ormedicaments. In addition PUFAs are commonly used in food, feed and inthe cosmetic industry. Poly unsaturated ω-3- and/or ω-6-fatty acids arean important part of animal feed and human food. Because of the commoncomposition of human food poly unsaturated ω-3-fatty acids, which are anessential component of fish oil, should be added to the food to increasethe nutritional value of the food; thus, for example, poly unsaturatedfatty acids such as docosahexaenoic acid or eicosapentaenoic acid areadded as mentioned above to infant formula to increase its nutritionalvalue.

Vitamins such as vitamin C, vitamin B12 or vitamin B2 are typicallyproduced with microorganism as mentioned above in the fermentationindustry. Vitamin C can be produced generally in a combined processusing biotransformation steps in combination with classical chemicalsynthesis. In another production process vitamin C is produced byfermentation alone. In general organisms such as Arthrobacter,Gluconobacter, Corynebacterium, Brevibacterium or Erwinia are used forvitamin C production. Vitamin B2 and vitamin B12 are produced withorganisms such as Bacillus, Streptomyces, Citrobacter, Klebsiella,Propionibacterium or Ashbya in large scale fermentation.

Commonly vitamin E and A are procuded in a classical chemical process orisolated from as natural vitamin E from plant oils. Vitamin E is animportant natural fat-soluble antioxidants. As such, vitamin E protectscell membranes from the damage caused by free radicals. High doses ofvitamin E have also been linked to a decreased ability of the blood toclot, which may be beneficial in those individuals at risk for heartdisease by reducing the risk of heart attack. A vitamin E deficiencyleads to pathophysiological situations in humans and animals. Of thedifferent types of vitamin E, the alpha tocopherol form is typicallyconsidered the “gold standard” in terms of antioxidant activity—althoughthe most recent research suggests that the other chemical forms maypossess equivalent or superior antioxidant protection. Vitamin Ecompounds therefore are of high economical value as additives in thefood and feed sectors, in pharmaceutical formulations and in cosmeticapplications. Vitamin A is another fat-soluble vitamin that is part of afamily of compounds including retinol, retinal and beta-carotene.Beta-carotene is also known as pro-vitamin A because it can be convertedinto vitamin A when additional levels are required. Vitamin A is neededby all of the body's tissues for general growth and repair processes andis especially important for bone formation, healthy skin/hair, nightvision and function of the immune system. Vitamin A may help boostimmune system function and resistance to infection. Vitamin Aderivatives are widely used in cosmetics and dermatological treatmentsfor skin preparations designed to combat skin aging and treat acne.Vitamin A has been used for decades as a treatment for variousvision-related conditions, including night blindness, cataracts,conjunctivitis, retinopathy and macular degeneration.

An economical method for producing of vitamins such as vitamin C, B2,B12 or vitamin E and food- and feedstuffs with increased vitamin contentare therefore very important. Particularly economical methods arebiotechnological methods utilizing vitamin-producing organisms, whichare either natural or optimized by genetic modification.

Carotenoids are a large family of compounds including over 600 memberssuch as β-carotene, lycopene or lutein. Carotenoids are widelydistributed in fruits and vegetables and are responsible, along withflavonoids, for contributing the color to many plants (a rule of thumbis the brighter, the better). In terms of nutrition, β-carotene'sprimary role is as mentioned above a precursor to vitamin A. β-caroteneas most other carotenoids, is a powerful antioxidant—so it has beenrecommended to protect against a variety of diseases such as cancer,cataracts and heart disease.

The introduction of a new gene or new genes for the synthesis of finechemicals into an organism or cell may not just increase thebiosynthetic flux towards an end product it may also increase or createde novo a new compound composition. Similarly, other genes involved inthe import of nutrients necessary for the biosynthesis of one or morefine chemicals (e.g., fatty acids, polar and neutral lipids, vitamins,enzymes etc.) may be increased in number or activity such that theseprecursors, cofactors, or intermediate compounds are increased inconcentration within the cell or within the storing compartment thusincreasing further the capability of the cell to produce the finechemical as described herein.

Amino acids are used in many branches of industry, including the food,animal feed, cosmetics, pharmaceutical and chemical industries. Aminoacids such as D,L-methionine, L-lysine or L-threonine are used in theanimal feed industry. The essential amino acids valine, leucine,isoleucine, lysine, threonine, methionine, tyrosine, phenylalanine andtryptophan are particularly important for the nutrition of mammalsespecially humans and a number of livestock species. Glycine,L-methionine and tryptophan are all used in the pharmaceutical industry.Glutamine, valine, leucine, isoleucine, histidine, arginine, proline,serine and alanine are used in the pharmaceutical and cosmeticsindustries. Threonine, tryptophan and D,L-methionine are widely usedfeed additives (Leuchtenberger, W. (1996) Amino acids—technicalproduction and use, pp. 466-502 in Rehm et al., (Ed.) Biotechnology vol.6, chapter 14a, VCH Weinheim). Moreover, amino acids are suitable forthe chemical industry as precursors for the synthesis of synthetic aminoacids and proteins, such as N-acetylcysteine,S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan and other subtancesdescribed in Ullmann's Encyclopedia of Industrial Chemistry, vol. A2,pp. 57-97, VCH Weinheim, 1985. To prefent physiological malnutritionsthe human body has a need for essentiall amino acids such as arginine,histidine, isoleucine, leucine, lysine, methionine, tyrosine,phenylalanine, threonine, tryptophan, and valine. Based on their contentof amino acids, foods are often classified as complete, partiallycomplete, or incomplete protein sources. In order for a protein to becomplete, it must contain all of the essential amino acids. This is thereason that many nutritionists rank non-meat foods as being incomplete.The foods do contain all amino acids, but some may be in lowerproportions than are required, and, therefore, should be combined withanother food containing higher amounts of these amino acids or should besupplemented with said essential amino acids.

Over one million tonnes of amino acids are currently produced annually;their market value amounts to over 2.5 billion US dollars. They arecurrently produced by four competing processes: Extraction from proteinhydrolysates, for example L-cystine, L-leucine or L-tyrosine, chemicalsynthesis, for example of D,L-methionine, conversion of chemicalprecursors in an enzyme or cell reactor, for example L-phenylalanine,and fermentative production by growing, on an industrial scale, bacteriawhich have been developed to produce and secrete large amounts of thedesired molecule in question. An organism, which is particularlysuitable for this purpose is Corynebacterium glutamicum, which is usedfor example for the production of L-lysine or L-glutamic acid. Otheramino acids which are produced by fermentation are, for example,L-threonine, L-tryptophan, L-aspartic acid and L-phenylalanine.

The biosynthesis of the natural amino acids in organisms capable ofproducing them, for example bacteria, has been characteriziedthoroughly; for a review of the bacterial amino acid biosynthesis andits regulation, see Umbarger, H. E. (1978) Ann. Rev. Biochem. 47:533-606].

It is known that amino acids are produced by fermentation of strains ofcoryneform bacteria, in particular Corynebacterium glutamicum. Due totheir great importance, the production processes are constantly beingimproved. Process improvements can relate to measures regardingtechnical aspects of the fermentation, such as, for example, stirringand oxygen supply, or the nutrient media composition, such as, forexample, the sugar concentration during fermentation, or to the work-upto give the product, for example by ion exchange chromatography, or tothe intrinsic performance properties of the microorganism itself.Bacteria from other genera such as Escherichia or Bacillus are also usedfor the production of amino acids. A number of mutant strains, whichproduce an assortment of desirable compounds from the group of thesulfur-containing fine chemicals have been developed via strainselection. The performance properties of said microorganisms areimproved with respect to the production of a particular molecule byapplying methods of mutagenesis, selection and mutant selection. Methodsfor the production of methionine have also been developed. In thismanner, strains are obtained which are, for example, resistant toantimetabolites, such as, for example, the methionine analoguesα-methylmethionine, ethionine, norleucine, N-acetylnorleucine,S-trifluoromethylhomocysteine, 2-amino-5-heprenoitic acid,selenomethionine, methionine sulfoximine, methoxine,1-aminocyclopentanecarboxylic acid or which are auxotrophic formetabolites with regulatory importance and which producesulfur-containing fine chemicals such as, for example, L-methionine.However, such processes developed for the production of methionine havethe disadvantage that their yields are too low for being economicallyexploitable and that they are therefore not yet competitive with regardto chemical synthesis.

Zeh (Plant Physiol., Vol. 127, 2001: 792-802) describes increasing themethionine content in potato plants by inhibiting threonine synthase bywhat is known as antisense technology. This leads to a reduced threoninesynthase activity without the threonine content in the plant beingreduced. This technology is highly complex; the enzymatic activity mustbe inhibited in a very differentiated manner since otherwiseauxotrophism for the amino acid occurs and the plant will no longergrow.

U.S. Pat. No. 5,589,616 teaches the production of higher amounts ofamino acids in plants by overexpressing a monocot storage protein indicots. WO 96/38574, WO 97/07665, WO 97/28247, U.S. Pat. No. 4,886,878,U.S. Pat. No. 5,082,993 and U.S. Pat. No. 5,670,635 are following thisapproach. That means in all the aforementioned intellectual propertyrights different proteins or polypeptides are expressed in plants. Saidproteins or polypeptides should function as amino acid sinks. Othermethods for increasing amino acids such as lysine are disclosed in WO95/15392, WO 96/38574, WO 89/11789 or WO 93/19190. In this casesspeziell enzymes in the amino acid biosynthetic pathway such as thediphydrodipicolinic acid synthase are deregulated. This leads to anincrease in the production of lysine in the different plants. Anotherapproach to increase the level of amino acids in plants is disclosed inEP-A-0 271 408. EP-A-0 271 408 teaches the mutagenensis of plant andselection afterwards with inhibitors of certain enzymes of amino acidbiosynthetic pathway.

Methods of recombinant DNA technology have also been used for some yearsto improve Corynebacterium strains producing L-amino acids by amplifyingindividual amino acid biosynthesis genes and investigating the effect onamino acid production.

As described above, the essential amino acids are necessary for humansand many mammals, for example for livestock. L-methionine is importantas methyl group donor for the biosynthesis of, for example, choline,creatine, adrenaline, bases and RNA and DNA, histidine, and for thetransmethylation following the formation of S-adenosylmethionine or as asulfhydryl group donor for the formation of cysteine. Moreover,L-methionine appears to have a positive effect in depression.

Improving the quality of foodstuffs and animal feeds is an importanttask of the food-and-feed industry. This is necessary since, forexample, certain amino acids, which occur in plants are limited withregard to the supply of mammals. Especially advantageous for the qualityof foodstuffs and animal feeds is as balanced as possible an amino acidprofile since a great excess of an amino acid above a specificconcentration in the food has no further positive effect on theutilization of the food since other amino acids suddenly becomelimiting. A further increase in quality is only possible via addition offurther amino acids, which are limiting under these conditions. Thetargeted addition of the limiting amino acid in the form of syntheticproducts must be carried out with extreme caution in order to avoidamino acid imbalance. For example, the addition of an essential aminoacid stimulates protein digestion, which may cause deficiency situationsfor the second or third limiting amino acid, in particular. In feedingexperiments, for example casein feeding experiments, the additionalprovision of methionine, which is limiting in casein, has revealed thefatty degeneration of liver, which could only be alleviated after theadditional provision of tryptophan.

To ensure a high quality of foods and animal feeds, it is thereforenecessary to add fine chemicals that means a plurality of compounds suchas amino acids, vitamins, organic acids, PUFAS etc. in a balanced mannerto suit the organism. Such supplemented food is named as “functionalfoods” or “nutraceuticals”. Nutraceuticals shall provide a healthbenefit to humans beyond basic nutrition. Functional foods havehealth-promoting or disease-preventing effects. Examples include omega-3fatty acids (found in many fish, flaxseed oil, soybean oil, canola oil,and walnuts), which reduce risk of coronary heart disease and lycopenein tomatoes, which has been associated with reduced risk of certaincancers.

From a practical standpoint it would be of great advantage to produce anorganism such as a microorganism or a plant containing a combination ofdifferent fine chemicals such as amino acids, vitamins, organic acids,carotenoids, PUFAS etc. at the same time in an sufficient amount toprovide optimal growth and health benefit to animals or humans insteadof combining different food or supplementing food or feed with differentfine chemicals.

It is therefore an object of the present invention to develop aninexpensive process for the synthesis of a combination of fine chemicalssuch as amino acids like tryptophane, proline, arginine, phenylalanine,tyrosine, alanine, glycine, threonine, serine, valine, isoleucine orleucine especially essential amino acids such as tryptophane, arginine,phenylalanine, tyrosine, threonine, valine, isoleucine or leucine,carbohydrates such as raffinose, maltose or inositol, vitamins such asγ-, α- or β-tocopherol, organic acids such as ferulic acid, malate orsinapic acid, carotenoids such as β-carotene etc. at the same time in ansufficient amount to provide optimal growth and health benefit toanimals or humans

It was now found that this object is achieved by providing the processaccording to the invention described herein and the embodimentscharacterized in the claims.

Accordingly, in a first embodiment, the invention relates to a processfor the production of a fine chemical, whereby the fine chemical is atleast one compound selected from the group consisting of tryptophane,proline, arginine, phenylalanine, tyrosine, alanine, glycine, threonine,serine, valine, isoleucine, leucine, raffinose, maltose, inositol,ferulic acid, malate, γ-tocopherol, α-tocopherol, β-tocopherol, ceroticacid, lignoceric acid, putrescine, sinapic acid,3,4-dihydroxyphenylaline (=DOPA), stearic acid and β-carotene.Accordingly, in the present invention, the term “the fine chemical” asused herein relates to an amino acid selected from the group consistingof tryptophane, proline, arginine, phenylalanine, tyrosine, alanine,glycine, threonine, serine, valine, isoleucine and leucine; a vitaminselected from the group consisting of γ-tocopherol, α-tocopherol andβ-tocopherol, a fatty acid selected from the group consisting of ceroticacid, lignoceric acid and stearic acid, an organic acid selected fromthe group consisting of ferulic acid, malate and sinapic acid or acompound selected from the group consisting of raffinose, putrescine,3,4-dihydroxyphenylaline (=DOPA) and β-carotene or mixtures thereofcontaining at least two, three, four or five compounds selected from theaforementioned groups, preferably 6, 7, 8 or 9 compounds selected fromthe aforementioned groups, most preferably 10, 11, 12, 13, 14, 15, 16,17 or more compounds selected from the aforementioned groups. Further,the term “the fine chemicals” as used herein also relates to finechemicals comprising at least one compound selected from the groupconsisting of tryptophane, proline, arginine, phenylalanine, tyrosine,alanine, glycine, threonine, serine, valine, isoleucine, leucine,raffinose, maltose, inositol, ferulic acid, malate, γ-tocopherol,α-tocopherol, β-tocopherol, cerotic acid, lignoceric acid, putrescine,sinapic acid, 3,4-dihydroxyphenylaline (=DOPA), stearic acid andβ-carotene.

In one embodiment, the term “the fine chemical” or “fine chemical” meansat least one compound selected from the group consisting ofL-tryptophane, L-proline, L-arginine, L-phenylalanine, L-tyrosine,L-alanine, glycine, L-threonine, L-serine, L-valine, L-isoleucine,L-leucine, raffinose, maltose, inositol, ferulic acid, malate,γ-tocopherol, α-tocopherol, β-tocopherol, cerotic acid, lignoceric acid,putrescine, sinapic acid, 3,4-dihydroxyphenylaline (=DOPA), stearic acidand β-carotene. Throughout the specification the term “the finechemical” means the aforementioned compounds, its salts, ester or amidsin free form or bound to other chemical compounds especially proteins.In a preferred embodiment, the term “the fine chemical” or “finechemical” means at least one compound selected from the group consistingof L-tryptophane, L-proline, L-arginine, L-phenylalanine, L-tyrosine,L-alanine, glycine, L-threonine, L-serine, L-valine, L-isoleucine andL-leucine in free form or its salts or bound to proteins.

Accordingly, the present invention relates to a process for theproduction of fine chemical comprising

-   (a) increasing or generating the biological activity represented by    a protein as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,    20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60,    62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,    96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,    124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,    150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,    176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,    202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,    228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,    254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,    280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,    306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,    332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,    358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,    384, 386, 388, 390, 392 or 394 in a non-human organism, or in one or    more parts thereof; and-   (b) growing the organism under conditions which permit the    production of the fine chemical in said organism.

Comprises/comprising and grammatical variations thereof when used inthis specification are to be taken to specify the presence of statedfeatures, integers, steps or components or groups thereof, but not topreclude the presence or addition of one or more other features,integers, steps, components or groups thereof.

Preferably, this process further comprises the step of recovering thefine chemical, which is synthesized by the organism from the organismand/or from the culture medium used for the growth or maintainance ofthe organism. The term “recovering” means the isolation of the finechemical in different purities, that means on the one hand harvesting ofthe biological material, which contains the fine chemical withoutfurther purification and on the other hand purities of the fine chemicalbetween 5% and 100% purity, preferred purities are in the range of 10%and 99%. In one embodiment, the purities are 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% or 99% or more.

Advantageously the process for the production of the fine chemical leadsto an enhanced production of the fine chemical. The terms “enhanced” or“increase” mean at least a 10%, 20%, 30%, 40% or 50%, preferably atleast 60%, 70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400%or 500% higher production of the fine chemical in comparison to thereference as defined below, e.g. that means in comparison to an organismwithout the aforementioned modification of the activity of a proteinhaving the biological activity represented by a protein as depicted inSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394.

Surprisingly it was found, that the transgenic expression of theKluyveromyces lactis, Cryptococcus neoformans, Neurospora crassa,Penicillium marneffei, Mucor rouxii, Schizophyllum commune,Paracoccidioides brasiliensis, Aspergillus fumigatus, Suillus bovinus,Candida albicans, Trichoderma reesei, Ashbya gossypii, Yarrowialipolytica, Ustilago maydis, Emericella nidulans, Trichomonas vaginalis,Colletotrichum trifolii, Blumeria graminis, Dictyostelium discoideum,Saccaromyces cerevisiae, Schizosaccharomyces pombe, Entamoebahistolytica, Oryza sativa, Brassica napus, Glycine max, Beta vulgaris,Lotus japonicus, Zinnia elegans, Zea mays, Cicer arietinum, Arabidopsisthaliana, Hordeum vulgare, Nicotiana tabacum, Gossypium hirsutum,Physcomitrella patens, Fucus distichus, Medicago truncutula, Homosapiens, Caenorhabditits elegans, Tigriopus japonicus, Rhopalosiphumpadi, Mus musculus, Discopyge ommata, Canis lupus, Drosophilamelanogaster, Anopheles gambiae, Aplysia california, Ciona savignyi,Ciona intestinalis, Hemicentrotus pulcherrimus, Giardia lamblia, Gallusgallus, Brachydanio rerio, Xenopus laevis, Xenopus tropicalis,Schistosoma japonicum, Schistosoma mansoni, Encephalitozoon cuniculi,Wuchereria bancrofti, Cavia porcellus, Sus scrofa, Rattus norvegicus,Pneumocystis carinii or Pagrus major proteins as depicted in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394for example in Arabidopsis thaliana conferred an increase in the finechemical content of the transformed plants.

In accordance with the invention, the term “organism” as understoodherein relates always to a non-human organism, in particular to ananimal or plant organism or to a microorganism. Further, the term“animal” as understood herein relates always to a non-human animal.

The sequence depicted in SEQ ID NO: 1 (YNL090W) from Saccharomycescerevisiae has been published in Madaule et al. [1987, “Characterizationof two members of the rho gene family from the yeast Saccharomycescerevisiae”; Proc. Natl. Acad. Sci., USA 84(3):779-83], and named asrho2. The gene encodes as all the other sequences mentioned above anduse in the inventive process a non-essential GTPase of the rho/racsubfamily of the ras-like GTPases. The protein may play a role in theestablishment of cell polarity or in microtubule assembly. Accordingly,in one embodiment, the process of the present invention comprises theuse of a gene product “involved in in the establishment of cell polarityor in microtubule assembly” from Saccaromyces cerevisiae or its homolog,e.g. as shown herein, for the production of the fine chemical, meaningof preferably for the production of essential amino acids, in particularfor increasing the amount of an essential amino acid in free or boundform in an organism or a part thereof, as mentioned herein. That meansthe increase of its biological activity by overexpression of theresponsible gene leads to an increase of the fine chemical.

The term “biological activity” means the biological function of theprotein of the invention. In contrast to the term “biological activity”the term “activity” means the increase in the production of the compoundproduced by the inventive process. The term “biological activity”preferably refers to for example the enzymatic function, transporter orcarrier function, DNA-packaging function, heat shock protein function,recombination protein function or regulatory function of a peptide orprotein in an organism, a tissue, a cell or a cell compartment. Suitablesubstrates are low-molecular-weight compounds and also the proteininteraction partners of a protein. The term “increase” of the biologicalfunction refers, for example, to the increase in binding capacity orbinding strength of a protein for at least one substrate in an organism,a tissue, a cell or a cell compartment—for example by one of the methodsdescribed herein below—in comparison with the wild type of the samegenus and species to which this method has not been applied, underotherwise identical conditions (such as, for example, cultureconditions, age of the plants and the like). Increase is also understoodas meaning the modification of the substrate specificity as can beexpressed for example, by the kcat/Km value. In this context, anincrease of the function of at least 10%, advantageously of at least20%, preferably at least 30%, especially preferably of at least 40%,50%, 60%, 70%, 80%, 90% or more, very especially preferably of at least150%, 200%, 250%, 300% or more, in comparison with the untreatedorganism is advantageous.

Homologues (=homologs) of the present gene products can be derived fromany organisms as long as the homologue confers the herein mentionedactivity, in particular, confers an increase in the fine chemical amountor content. Further, in the present invention, the term “homologue”relates to the sequence of an organism having the highest sequencehomology to the herein mentioned or listed sequences of all expressedsequences of said organism. However, the person skilled in the artknows, that preferably, the homologue has said the fine chemicalincreasing activity and, if known, the same biological function oractivity in the organism as the YNL090W protein as depicted in SEQ IDNO: 2. In one embodiment, the homolog of the SEQ ID NO: 2 is a homologhaving said biological activity and being derived from Eukaryot such asplants like the families Anacardiaceae, Asteraceae, Apiaceae,Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Caricaceae,Cannabaceae, Convolvulaceae, Chenopodiaceae, Cucurbitaceae,Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae,Gramineae, Juglandaceae, Lauraceae, Leguminosae or Linaceae; algae,fungi or mosses. In another embodiment, the homolog of SEQ ID NO: 2 is ahomolog having said activity and being derived from bacteria. In afurther embodiment, the homolog of the SEQ ID NO: 2 is a homolog havingsaid activity and being derived from fungi. I

Further homologs of are described herein below.

In accordance with the invention, a protein or polypeptide has the “thebiological activity represented by a protein as depicted in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394”if its de novo activity, or its increased expression directly orindirectly leads to an increased the fine chemical level in the organismor a part thereof, preferably in a cell of said organism and the proteinhas the above mentioned activities of a protein as depicted in SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308,310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or394. During the specification the activity or preferably the biologicalactivity of such a protein or polypeptide or an nucleic acid molecule orsequence encoding such protein or polypeptide is identical or similar ifit still has the biological or enzymatic activity of a protein asdepicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300,302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328,330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,386, 388, 390, 392 or 394, if it has at least 10% of the originalbiological or enzymatic activity, preferably 20%, particularlypreferably 30%, most particularly preferably 40% in comparison to aprotein as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392 or 394 of Kluyveromyces lactis,Cryptococcus neoformans, Neurospora crassa, Penicillium marneffei, Mucorrouxii, Schizophyllum commune, Paracoccidioides brasiliensis,Aspergillus fumigatus, Suillus bovinus, Candida albicans, Trichodermareesei, Ashbya gossypii, Yarrowia lipolytica, Ustilago maydis,Emericella nidulans, Trichomonas vaginalis, Colletotrichum trifolii,Blumeria graminis, Dictyostelium discoideum, Saccaromyces cerevisiae,Schizosaccharomyces pombe, Entamoeba histolytica, Oryza sativa, Brassicanapus, Glycine max, Beta vulgaris, Lotus japonicus, Zinnia elegans, Zeamays, Cicer arietinum, Arabidopsis thaliana, Hordeum vulgare, Nicotianatabacum, Gossypium hirsutum, Physcomitrella patens, Fucus distichus,Medicago truncutula, Homo sapiens, Caenorhabditits elegans, Tigriopusjaponicus, Rhopalosiphum padi, Mus musculus, Discopyge ommata, Canislupus, Drosophila melanogaster, Anopheles gambiae, Aplysia california,Ciona savignyi, Ciona intestinalis, Hemicentrotus pulcherrimus, Giardialamblia, Gallus gallus, Brachydanio rerio, Xenopus laevis, Xenopustropicalis, Schistosoma japonicum, Schistosoma mansoni, Encephalitozooncuniculi, Wuchereria bancrofti, Cavia porcellus, Sus scrofa and/orPagrus major.

The terms “increased”, “rised”, “extended”, “enhanced”, “improved” or“amplified” relate to a corresponding change of a property in anorganism, a part of an organism such as a tissue, seed, root, leave,flower etc. or in a cell and are interchangeable. Preferably, theoverall activity in the volume is increased or enhanced in cases if theincrease or enhancement is related to the increase or enhancement of anactivity of a gene product, independent whether the amount of geneproduct or the specific activity of the gene product or both isincreased or enhanced or whether the amount, stability or translationefficacy of the nucleic acid sequence or gene encoding for the geneproduct is increased or enhanced. The terms “reduction”, “decrease” or“deletion” relate to a corresponding change of a property in, anorganism, a part of an organism such as a tissue, seed, root, leave,flower etc. or in a cell. Preferably, the overall activity in the volumeis reduced, decreased or deleted in cases if the reduction, decrease ordeletion is related to the reduction, decrease or deletion of anactivity of a gene product, independent whether the amount of geneproduct or the specific activity of the gene product or both is reduced,decreased or deleted or whether the amount, stability or translationefficacy of the nucleic acid sequence or gene encoding for the geneproduct is reduced, decreased or deleted.

The terms “increase” or “decrease” relate to a corresponding change of aproperty an organism or in a part of an organism, such as a tissue,seed, root, leave, flower etc. or in a cell. Preferably, the overallactivity in the volume is increased in cases the increase relates to theincrease of an activity of a gene product, independent whether theamount of gene product or the specific activity of the gene product orboth is increased or generated or whether the amount, stability ortranslation efficacy of the nucleic acid sequence or gene encoding forthe gene product is increased.

Under “change of a property” it is understood that the activity,expression level or amount of a gene product or the metabolite contentis changed in a specific volume relative to a corresponding volume of acontrol, reference or wild type, including the de novo creation of theactivity or expression.

The terms “increase” or “decrease” include the change of said propertyin only parts of the subject of the present invention, for example, themodification can be found in compartment of a cell, like a organelle, orin a part of a plant, like tissue, seed, root, leave, flower etc. but isnot detectable if the overall subject, i.e. complete cell or plant, istested. Preferably, the increase or decrease is found cellular, thus theterm “increase of an activity” or “increase of a metabolite content”relates to the cellular increase compared to the wild typ cell.Typically said increase is based on the higher content of thetranscribed nucleic acid or the protein translated from said nucleicacid sequence in the modified cell in comparison to the wild type cell.

Accordingly, the term “increase” or “decrease” means that the specificactivity of an enzyme as well as the amount of a compound or metabolite,e.g. of a polypeptide, a nucleic acid molelcule or of the fine chemicalof the invention or an encoding mRNA or DNA, can be increased ordecreased in a volume.

The terms “wild type”, “control” or “reference” are exchangeable and canbe a cell or a part of organisms such as an organelle or a tissue, or anorganism, in particular a microorganism or a plant, which was notmodified or treated according to the herein described process accordingto the invention. Accordingly, the cell or a part of organisms such asan organelle or a tissue, or an organism, in particular a microorganismor a plant used as wild typ, control or reference corresponds to thecell, organism or part thereof as much as possible and is in any otherproperty but in the result of the process of the invention as identicalto the subject matter of the invention as possible. Thus, the wild type,control or reference is treated identically or as identical as possible,saying that only conditions or properties might be different which donot influence the quality of the tested property.

Preferably, any comparison is carried out under analogous conditions.The term “analogous conditions” means that all conditions such as, forexample, culture or growing conditions, assay conditions (such as buffercomposition, temperature, substrates, pathogen strain, concentrationsand the like) are kept identical between the experiments to be compared.

The “reference”, “control”, or “wild type” is preferably a subject, e.g.an organelle, a cell, a tissue, an organism, in particular a plant or amicroorganism, which was not modified or treated according to the hereindescribed process of the invention and is in any other property assimilar to the subject matter of the invention as possible. Thereference, control or wild type is in its genome, transcriptome,proteome or meta-bolome as similar as possible to the subject of thepresent invention. Preferably, the term “reference-” “control-” or “wildtype-”-organelle, -cell, -tissue or -organism, in particular plant ormicroorganism, relates to an organelle, cell, tissue or organism, inparticular plant or micororganism, which is nearly genetically identicalto the organelle, cell, tissue or organism, in particular microorganismor plant, of the present invention or a part thereof preferably 95%,more peferred are 98%, even more preferred are 99.00%, in particular99.10%, 99.30%, 99.50%, 99.70%, 99.90%, 99.99%, 99.999% or more. Mostpreferable the “reference”, “control”, or “wild type” is a subject, e.g.an organelle, a cell, a tissue, an organism, which is geneticallyidentical to the organism, cell or organelle used according to theprocess of the invention except that the responsible or acvitivityconferring nucleic acid molecules or the gene product encoded by themare amended, manipulated, exchanged or introduced according to theinventive process.

Preferably, the reference, control or wild type differs form the subjectof the present invention only in the cellular activity of thepolypeptide of the invention, e.g. as result of an increase in the levelof the nucleic acid molecule of the present invention or an increase ofthe specific activity of the polypeptide of the invention, e.g. by or inthe expression level or activity of an protein having the biologicalactivity represented by a protein as depicted in SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370,372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 or itshomologs, its biochemical or genetical causes and the increased amountof the fine chemical.

In case, a control, reference or wild type differing from the subject ofthe present invention only by not being subject of the process of theinvention can not be provided, a control, reference or wild type can bean organism in which the cause for the modulation of an activityconferring the increase of the fine chemical or expression of thenucleic acid molecule of the invention as described herein has beenswitched back or off, e.g. by knocking out the expression of responsiblegene product, e.g. by antisense inhibition, by inactivation of anactivator or agonist, by activation of an inhibitor or antagonist, byinhibition through adding inhibitory antibodies, by adding activecompounds as e.g. hormones, by introducing negative dominant mutants,etc. A gene production can for example be knocked out by introducinginactivating point mutations, which lead to an enzymatic activityinhibition or a destabilization or an inhibition of the ability to bindto cofactors etc.

Accordingly, preferred reference subject is the starting subject of thepresent process of the invention. Preferably, the reference and thesubject matter of the invention are compared after standardization andnormalization, e.g. to the amount of total RNA, DNA, or protein oractivity or expression of reference genes, like housekeeping genes, suchas ubiquitin, actin or ribosomal proteins.

A series of mechanisms exists via which a modification of the a protein,e.g. the polypeptide of the invention can directly or indirectly affectthe yield, production and/or production efficiency of the amino acid.

For example, the molecule number or the specific activity of thepolypeptide or the nucleic acid molecule may be increased. Largeramounts of the fine chemical can be produced if the polypeptide or thenucleic acid of the invention is expressed de novo in an organismlacking the activity of said protein. However, it is also possible toincrease the expression of the gene which is naturally present in theorganisms, for example by modifying the regulation of the gene, or byincreasing the stability of the corresponding mRNA or of thecorresponding gene product encoded by the nucleic acid molecule of theinvention, or by introducing homologous genes from other organisms whichare differently regulated, eg. not feedback sensitive or not feedbackregulated.

This also applies analogously to the combined increased expression ofthe nucleic acid molecule of the present invention or its gene productwith that of further enzymes of the biochemical pathway of the finechemical e.g. of the amino acid biosynthesis pathway, e.g. which areuseful for the synthesis of the fine chemicals.

The increase, decrease or modulation according to this invention can beconstitutive, e.g. due to a stable permanent transgenic expression or toa stable mutation in the corresponding endogenous gene encoding thenucleic acid molecule of the invention or to a modulation of theexpression or of the behaviour of a gene conferring the expression ofthe polypeptide of the invention, or transient, e.g. due to an transienttransformation or temporary addition of a modulator such as a agonist orantagonist or inducible, e.g. after transformation with a inducibleconstruct carrying the nucleic acid molecule of the invention undercontrol of a induceable promoter and adding the inducer, e.g.tetracycline or as described herein below.

The increase in activity of the polypeptide amounts in a cell, a tissue,a organelle, an organ or an organism or a part thereof preferably to atleast 5%, preferably to at least 20% or at to least 50%, especiallypreferably to at least 70%, 80%, 90% or more, very especially preferablyare to at least 200%, 300% or 400%, most preferably are to at least500%, 600% or more in comparison to the control, reference or wild type.

The specific activity of a polypeptide encoded by a nucleic acidmolecule of the present invention or of the polypeptide of the presentinvention can be tested as described in the examples. In particular, theexpression of a protein in question in a cell, e.g. a plant cell or amicroorganism and the detection of an increase the fine chemical levelin comparison to a control is an easy test and can be performed asdescribed in the state of the art.

The term “increase” includes, that a compound or an activity isintroduced into a cell de novo or that the compound or the activity hasnot been detectable before, in other words it is “generated”.

Accordingly, in the following, the term “increasing” also comprises theterm “generating” or “stimulating”. The increased activity manifestsitself in an increase of the fine chemical.

In case the biological activity of the Saccaromyces cerevisiae proteinYNL090W as depicted in SEQ ID NO: 2, or the biological activity of theof Kluyveromyces lactis, Cryptococcus neoformans, Neurospora crassa,Penicillium marneffei, Mucor rouxii, Schizophyllum commune,Paracoccidioides brasiliensis, Aspergillus fumigatus, Suillus bovinus,Candida albicans, Trichoderma reesei, Ashbya gossypii, Yarrowialipolytica, Ustilago maydis, Emericella nidulans, Trichomonas vaginalis,Colletotrichum trifolii, Blumeria graminis, Dictyostelium discoideum,Schizosaccharomyces pombe, Entamoeba histolytica, Oryza sativa, Brassicanapus, Glycine max, Beta vulgaris, Lotus japonicus, Zinnia elegans, Zeamays, Cicer arietinum, Arabidopsis thaliana, Hordeum vulgare, Nicotianatabacum, Gossypium hirsutum, Physcomitrella patens, Fucus distichus,Medicago truncutula, Homo sapiens, Caenorhabditits elegans, Tigriopusjaponicus, Rhopalosiphum padi, Mus musculus, Discopyge ommata, Canislupus, Drosophila melanogaster, Anopheles gambiae, Aplysia california,Ciona savignyi, Ciona intestinalis, Hemicentrotus pulcherrimus, Giardialamblia, Gallus gallus, Brachydanio rerio, Xenopus laevis, Xenopustropicalis, Schistosoma japonicum, Schistosoma mansoni, Encephalitozooncuniculi, Wuchereria bancrofti, Cavia porcellus, Sus scrofa, Rattusnorvegicus, Pneumocystis carinii or Pagrus major proteins as depictedSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306,308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334,336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362,364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,392 or 394 or its homologs is increased, preferably, in one embodment anincrease of the fine chemical of at least 100%, 150%, or preferably toat least 200%, 250%, 300%, 350% or 400%, especially prerferably to atleast 450%, 500%, 550%, 600% or more is conferred.

In case the biological activity of the Saccaromyces cerevisiae proteinYNL090W as depicted in SEQ ID NO: 2, or the biological activity of theKluyveromyces lactis, Cryptococcus neoformans, Neurospora crassa,Penicillium marneffei, Mucor rouxii, Schizophyllum commune,Paracoccidioides brasiliensis, Aspergillus fumigatus, Suillus bovinus,Candida albicans, Trichoderma reesei, Ashbya gossypii, Yarrowialipolytica, Ustilago maydis, Emericella nidulans, Trichomonas vaginalis,Colletotrichum trifolii, Blumeria graminis, Dictyostelium discoideum,Schizosaccharomyces pombe, Entamoeba histolytica, Oryza sativa, Brassicanapus, Glycine max, Beta vulgaris, Lotus japonicus, Zinnia elegans, Zeamays, Cicer arietinum, Arabidopsis thaliana, Hordeum vulgare, Nicotianatabacum, Gossypium hirsutum, Physcomitrella patens, Fucus distichus,Medicago truncutula, Homo sapiens, Caenorhabditits elegans, Tigriopusjaponicus, Rhopalosiphum padi, Mus musculus, Discopyge ommata, Canislupus, Drosophila melanogaster, Anopheles gambiae, Aplysia califomia,Ciona savignyi, Ciona intestinalis, Hemicentrotus pulcherrimus, Giardialamblia, Gallus gallus, Brachydanio rerio, Xenopus laevis, Xenopustropicalis, Schistosoma japonicum, Schistosoma mansoni, Encephalitozooncuniculi, Wuchereria bancrofti, Cavia porcellus, Sus scrofa, Rattusnorvegicus, Pneumocystis carinii or Pagrus major proteins as depictedSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306,308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334,336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362,364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,392 or 394 or its homologs is increased, preferably, an increase of thefine chemical in conferred, preferably of the fine chemical such asessential amino acids e.g. tryptophane, arginine, phenylalanine,tyrosine, threonine, valine, isoleucine and/or leucine, non-essentialamino acids e.g. proline, alanine, glycine or serine, modified aminoacids e.g. 3,4-dihydroxyphenylalanine, carbohydrates e.g. raffinose,inositol or iso-maltose, vitamins e.g. α-tocopherol, β-tocopherol orγ-tocopherol, organic acids e.g. ferulic acid, sinapic acid or malate,fatty acids e.g. cerotic acid, lignoceric acid, 2-hydroxy-palmitic acidor stearic acid, carotinoids e.g. β-carotene or mixtures thereof isconferred.

In this context, the fine chemical amount in a cell, preferably in atissue, more preferred in a organism as a plant or a microorganim orpart thereof, is increased by at least 3%, 4%, 5%, 6%, 7%, 8% or 9% ormore, especially preferably are at least 10%, 20%, 40%, 50% or more,very especially preferably are more than 60%, 70%, 80%, 90%, 100% ormore and most preferably are 150% or more, such as 200%, 250%, 300%,350%, 400%, 450%, 500%, 550% or 600%.

The fine chemical can be contained in the organism either in its freeform and/or bound to proteins, polypeptids or other compounds such aspolysaccharides, lipids, glycoproteins or glycolipids etc. or mixturesthereof. Accordingly, in one embodiment, the amount of the free form ina cell, preferably in a tissue, more preferred in a organism as a plantor a microorganim or part thereof, is increased by at least 3%, 4%, 5%,6%, 7%, 8% or 9% or more, especially preferably are at least 10%, 20%,40%, 50% or more, very especially preferably are more than 60%, 70%,80%, 90%, 100% or more and most preferably are 150% or more, such as200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or 600%. Accordingly, inan other embodiment, the amount of the bound the fine chemical in acell, preferably in a tissue, more preferred in a organism as a plant ora microorganism or part thereof, is increased by at least 3%, 4%, 5%,6%, 7%, 8% or 9% or more, especially preferably are at least 10%, 20%,40%, 50% or more, very especially preferably are more than 60%, 70%,80%, 90%, 100% or more and most preferably are 150% or more, such as200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or 600%.

A protein having an activity conferring an increase in the amount orlevel of the fine chemical preferably has the structure of thepolypeptide described herein, in particular of the polypeptidescomprising the consensus sequence shown in SEQ ID NO: 47, SEQ ID NO: 48,SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:397, SEQ ID NO: 398, SEQ ID NO: 399 and/or SEQ ID NO: 400 as describedherein, or is encoded by the nucleic acid molecule characterized hereinor the nucleic acid molecule according to the invention, for example bythe nucleic acid molecule as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179,181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291,293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319,321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,377, 379, 381, 383, 385, 387, 389, 391 or 393 or its herein describedfunctional homologues and has the herein mentioned activity.

For the purposes of the present invention, the terms “the fine chemical”or “fine chemical” such as essential amino acids e.g. tryptophane,arginine, phenylalanine, tyrosine, threonine, valine, isoleucine and/orleucine, non-essential amino acids e.g. proline, alanine, glycine orserine, modified amino acids e.g. 3,4-dihydroxyphenylalanine,carbohydrates e.g. raffinose, inositol or iso-maltose, vitamins e.g.α-tocopherol, β-tocopherol or γ-tocopherol, organic acids e.g. ferulicacid, sinapic acid or malate, fatty acids e.g. cerotic acid, lignocericacid, 2-hydroxy-palmitic acid or stearic acid, carotinoids e.g.β-carotene or mixtures thereof also encompass the corresponding salts,such as, for example, tryptophane hydrochloride, arginine hydrochloride,phenylalanine hydrochloride, tyrosine hydrochloride, threoninehydrochloride, valine hydrochloride, isoleucine hydrochloride or leucinehydrochloride or tryptophane sulfate, arginine sulfate, phenylalaninesulfate, tyrosine sulfate, threonine sulfate, valine sulfate, isoleucinesulfate or leucine sulfate; ester or amids.

Owing to the biological activity of the proteins which are used in theprocess according to the invention and which are encoded by nucleic acidmolecules according to the invention, it is possible to producecompositions comprising the fine chemical, i.e. an increased amount ofthe free chemical free or bound, e.g amino acid compositions. Dependingon the choice of the organism used for the process according to thepresent invention, for example a microorganism or a plant, compositionsor mixtures of various fine chemicals e.g. amino acids can be produced.

The term “expression” refers to the transcription and/or translation ofa codogenic gene segment or gene. As a rule, the resulting product is anmRNA or a protein. However, expression products can also includefunctional RNAs such as, for example, antisense, nucleic acids, tRNAs,snRNAs, rRNAs, RNAi, siRNA, ribozymes etc. Expression may be systemic,local or temporal, for example limited to certain cell types,tissuesorgans or time periods.

In one embodiment, the process of the present invention comprises one ormore of the following steps:

-   a) stabilizing a protein conferring the increased expression of a    protein encoded by the nucleic acid molecule of the invention or of    the protein of the invention, e.g. of a protein having the    biological activity represented by a protein as depicted in SEQ ID    NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,    36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,    78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,    110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,    136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,    162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,    188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,    214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,    240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,    266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,    292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,    318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,    344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,    370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 or    its homologs having the fine chemical increasing activity;-   b) stabilizing a mRNA conferring the increased expression of a    protein encoded by the nucleic acid molecule of the invention, e.g.    of a protein having the biological activity represented by a protein    as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,    24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,    66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,    100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,    126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,    152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,    178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,    204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,    230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,    256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,    282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306,    308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,    334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,    360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,    386, 388, 390, 392 or 394 or its homologs or of an mRNA encoding the    polypeptide of the present invention having the fine chemical    increasing activity;-   c) increasing the specific activity of a protein conferring the    increased expression of a protein encoded by the nucleic acid    molecule of the invention or of the protein of the invention having    the fine chemical increasing activity, e.g. of a protein having the    biological activity represented by a protein as depicted in SEQ ID    NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,    36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,    78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,    110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,    136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,    162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,    188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,    214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,    240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,    266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,    292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,    318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,    344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,    370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 or    its homologs, or decreasing the inhibitiory regulation of the    protein of the invention;-   d) generating or increasing the expression of an endogenous or    artificial transcription factor mediating the expression of a    protein conferring the increased expression of a protein encoded by    the nucleic acid molecule of the invention or of the protein of the    invention having the fine chemical increasing activity, e.g. of a    polypeptide having the biological activity represented by a protein    as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,    24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,    66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,    100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,    126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,    152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,    178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,    204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,    230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,    256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,    282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306,    308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,    334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,    360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,    386, 388, 390, 392 or 394 or its homologs;-   e) stimulating activity of a protein conferring the increased    expression of a protein encoded by the nucleic acid molecule of the    present invention or a protein of the present invention having the    fine chemical increasing activity, e.g. of a protein having the    biological activity represented by a protein as depicted in SEQ ID    NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,    36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,    78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,    110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,    136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,    162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,    188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,    214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,    240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,    266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,    292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,    318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,    344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,    370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 or    its homologs, by adding one or more exogenous inducing factors to    the organismus or parts thereof;-   f) expressing a transgenic gene encoding a protein conferring the    increased expression of a polypeptide (=protein) encoded by the    nucleic acid molecule or the protein of the invention, having the    fine chemical increasing activity, e.g. of a protein having the    biological activity represented by a protein as depicted in SEQ ID    NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,    36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,    78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,    110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,    136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,    162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,    188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,    214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,    240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,    266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,    292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,    318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,    344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,    370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 or    its homologs;-   g) increasing the copy number of a gene conferring the increased    expression of a nucleic acid molecule encoding a protein encoded by    the nucleic acid molecule of the invention or the protein of the    invention the fine chemical increasing activity, e.g. of a protein    having the biological activity represented by a protein as depicted    in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,    30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70,    72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,    104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,    130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,    156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,    182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,    208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,    234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,    260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,    286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,    312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336,    338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362,    364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,    390, 392 or 394 or its homologs;-   h) increasing the expression of the endogenous gene encoding the    protein of the invention, e.g. a protein having the biological    activity represented by a protein as depicted in SEQ ID NO: 2, 4, 6,    8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,    42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,    84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,    114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,    140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,    166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,    192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,    218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,    244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,    270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,    296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,    322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,    348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372,    374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 or its    homologs, by adding positive expression or removing negative    expression elements, e.g. homologous recombination can be used to    either introduce positive regulatory elements like for plants the    35S enhancer into the promoter or to remove repressor elements form    regulatory regions. Further gene conversion methods can be used to    disrupt repressor elements or to enhance to acitivty of positive    elements. Positive elements can be randomly introduced in plants by    T-DNA or transposon mutagenesis and lines can be identified in which    the positive elements have be integrated near to a gene of the    invention, the expression of which is thereby enhanced;-   i) modulating growth conditions of an organism in such a manner,    that the expression or activity of the gene encoding the protein of    the invention or the protein itself is enhanced for example    microorganisms or plants can be grown under a higher temperature    regime leading to an enhanced expression of heat shock proteins,    e.g. the heat shock protein of the invention, which can lead an    enhanced the fine chemical production; and/or-   j) selecting of organisms with expecially high activity of the    protein of the invention from natural or from mutagenized resources    and breeding them into the target organisms, eg the elite crops.

Preferably, said mRNA is the nucleic acid molecule of the inventionand/or the protein conferring the increased expression of a proteinencoded by the nucleic acid molecule of the invention or the polypeptidehaving the herein mentioned activity, e.g. conferring the increase ofthe fine chemical after increasing the expression or activity of theencoded polypeptide or having the activity of a polypeptide havingbiological activity represented by a protein as depicted in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394or its homologs.

In general, the amount of mRNA, polynucleotide or nucleic acid moleculein a cell or a compartment of an organism correlates with the amount ofencoded protein and thus with the overall activity of the encodedprotein in said volume. Said correlation is not always linear, theactivity in the volume is dependent on the stability of the molecules,the degradation of the molecules or the presence of activating orinhibiting co-factors. Further, product and educt inhibitions of enzymesare well known and described in textbooks, e.g. Stryer, Biochemistry orZinser et al. “Enzyminhibitoren”/Enzyme inhibitors”.

The activity of the abovementioned proteins and/or poylpeptide encodedby the nucleic acid molecule of the present invention can be increasedin various ways. For example, the activity in an organism or in a partthereof, like a cell, is increased via increasing the gene productnumber, e.g. by increasing the expression rate, like introducing astronger promoter, or by increasing the stability of the mRNA expressed,thus increasing the translation rate, and/or increasing the stability ofthe gene product, thus reducing the proteins decayed. Further, theactivity or turnover of enzymes can be influenced in such a way that areduction or increase of the reaction rate or a modification (reductionor increase) of the affinity to the substrate results, is reached. Amutation in the catalytic centre of a polypeptide of the invention, e.g.an enzyme, can modulate the turn over rate of the enzyme, e.g. a knockout of an essential amino acid can lead to a reduced or completely knockout activity of the enzyme, or the deletion or mutation of regulatorbinding sites can reduce a negative regulation like a feedbackinhibition (or a substrate inhibition, if the substrate level is alsoincreased). The specific activity of an enzyme of the present inventioncan be increased such that the turn over rate is increased or thebinding of a co-factor is improved. Improving the stability of theencoding mRNA or the protein can also increase the activity of a geneproduct. The stimulation of the activity is also under the scope of theterm “increased activity”.

Moreover, the regulation of the abovementioned nucleic acid sequencesmay be modified so that gene expression is increased. This can beachieved advantageously by means of heterologous regulatory sequences orby modifying, for example mutating, the natural regulatory sequenceswhich are present. The advantageous methods may also be combined witheach other.

In general, an activity of a gene product in an organism or partthereof, in particular in a plant cell, a plant, or a plant tissue or apart thereof or in a microorganism can be increased by increasing theamount of the specific encoding mRNA or the corresponding protein insaid organism or part thereof. “Amount of protein or mRNA” is understoodas meaning the molecule number of polypeptides or mRNA molecules in anorganism, a tissue, a cell or a cell compartment. “Increase” in theamount of a protein means the quantitative increase of the moleculenumber of said protein in an organism, a tissue, a cell or a cellcompartment or part thereof—for example by one of the methods describedherein below—in comparison to a wild type, control or reference.

The increase in molecule number amounts preferably to at least1%,preferably to more than 10%, more preferably to 30% or more, especiallypreferably to 50%, 70% or more, very especially preferably to 100%, mostpreferably to 500% or more. However, a de novo expression is alsoregarded as subject of the present invention.

A modification, i.e. an increase or decrease, can be caused byendogenous or exogenous factors. For example, an increase in activity inan organism or a part thereof can be caused by adding a gene product ora precursor or an activator or an agonist to the media or nutrition orcan be caused by introducing said subjects into a organism, transient orstable.

In one embodiment the increase in the amount of the fine chemical in theorganism or a part thereof, e.g. in a cell, a tissue, an organ, anorganelle etc., is achived by increasing the endogenous level of thepolypeptide of the invention. Accordingly, in an embodiment of thepresent invention, the present invention relates to a process whereinthe gene copy number of a gene encoding the polynucleotide or nucleicacid molecule of the invention is increased. Further, the endogenouslevel of the polypeptide of the invention can for example be increasedby modifiying the transcriptional or translational regulation of thepolypeptide.

In one embodiment the amount of the fine chemical in the organism orpart thereof can be increase by targeted or random mutagenesis of theendogenous genes of the invention. For example homologous recombinationcan be used to either introduce positive regulatory elements like forplants the 35S enhancer into the promoter or to remove repressorelements form regulatory regions. In addition gene conversion likemethods described by Kochevenko and Willmitzer (Plant Physiol. 2003 May;132(1): 174-84) and citations therein can be used to disrupt repressorelements or to enhance to acitivty of positive regulatory elements.Furthermore positive elements can be randomly introduced in (plant)genomes by T-DNA or transposon mutagenesis and lines can be screenedfor, in which the positive elements has be integrated near to a gene ofthe invention, the expression of which is thereby enhanced. Theactivation of plant genes by random integrations of enhancer elementshas been described by Hayashi et al., 1992 (Science 258:1350-1353) orWeigel et al., 2000 (Plant Physiol. 122, 1003-1013) and others citatedtherein. Reverse genetic strategies to identify insertions (whicheventually carrying the activation elements) near in genes of interesthave been described for various cases eg. Krysan et al., 1999 (PlantCell 1999, 11, 2283-2290); Sessions et al., 2002 (Plant Cell 2002, 14,2985-2994); Young et al., 2001, (Plant Physiol. 2001, 125, 513-518);Koprek et al., 2000 (Plant J. 2000, 24, 253-263); Jeon et al., 2000(Plant J. 2000, 22, 561-570); Tissier et al., 1999 (Plant Cell 1999, 11,1841-1852); Speulmann et al., 1999 (Plant Cell 1999, 11, 1853-1866).Briefly material from all plants of a large T-DNA or transposonmutagenized plant population is harvested and genomic DNA prepared. Thenthe genomic DNA is pooled following specific architectures as describedfor example in Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290).Pools of genomics DNAs are then screened by specific multiplex PCRreactions detecting the combination of the insertional mutagen (eg T-DNAor Transposon) and the gene of interest. Therefore PCR reactions are runon the DNA pools with specific combinations of T-DNA or transposonborder primers and gene specific primers. General rules for primerdesign can again be taken from Krysan et al., 1999 (Plant Cell 1999, 11,2283-2290) Rescreening of lower levels DNA pools lead to theidentifcation of individual plants in which the gene of interest isdisrupted by the insertional mutagen. The enhancement of positiveregulatory elements or the disruption or weaking of negative regulatoryelements can also be achieved through common mutagenesis techniques: Theproduction of chemically or radiation mutated populations is a commontechnique and known to the skilled worker. Methods for plants aredescribed by Koorneef et al. 1982 and the citations therein and byLightner and Caspar in “Methods in Molecular Biology” Vol 82. Thesetechniques usually induce pointmutations that can be identified in anyknown gene using methods such as TILLING (Colbert et al. 2001).

Accordingly, the expression level can be increased if the endogenousgenes encoding a polypeptide conferring an increased expression of thepolypeptide of the present invention, in particular genes comprising thenucleic acid molecule of the present invention, are modified viahomologous recombination, Tilling approaches or gene conversion

Regulatory sequences can be operatively linked to the coding region ofan endogenous protein and control its transcription and translation orthe stability or decay of the encoding mRNA or the expressed protein. Inorder to modify and control the expression, promoter, UTRs, splicingsites, processing signals, polyadenylation sites, terminators,enhancers, repressors, post transcriptional or posttranslationalmodification sites can be changed, added or amended for example, theactivation of plant genes by random integrations of enhancer elementshas been described by Hayashi et al., 1992 (Science 258:1350-1353) orWeigel et al., 2000 (Plant Physiol. 122, 1003-1013) and others citatedtherein. For example, the expression level of the endogenous protein canbe modulated by replacing the endogenous promoter with a strongertransgenic promoter or by replacing the endogenous 3′UTR with a 3′UTR,which provides more stablitiy without amending the coding region.Further, the transcriptional regulation can be modulated by introductionof an artifical transcription factor as described in the examples.Alternative promoters, terminators and UTR are described below.

The activation of an endogenous polypeptide having the fine chemicalincreasing activity, e.g. having the biological activity represented bya protein as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392 or 394, e.g. conferring the increase of thefine chemical after increase of expression or activity can also beincreased by introducing a synthetic transcription factor, which bindsclose to the coding region of the protein of the invention encoding geneand activates its transcription. A chimeric zinc finger protein can beconstrued, which comprises a specific DNA-binding domain and anactivation domain as e.g. the VP16 domain of Herpes Simplex virus. Thespecific binding domain can bind to the regulatory region of the nucleicacid sequence used in the inventive process. The expression of thechimeric transcription factor in an organism, in particular in a plant,leads to a specific expression of the protein of the invention, see e.g.in WO01/52620, Oriz, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, 13290 orGuan, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, 13296.

In one further embodiment of the process according to the invention,organisms are used in which one of the abovementioned genes, or one ofthe abovementioned nucleic acids, is mutated in a way that the activityof the encoded gene products is less influenced by cellular factors, ornot at all, in comparison with the unmutated proteins. For example, wellknown regulation mechanism of enzymic activity are substrate inhibitionor feed back regulation mechanisms. Ways and techniques for theintroduction of substitutions, deletions and additions of one or morebases, nucleotides or amino acids of a corresponding sequence aredescribed herein below in the corresponding paragraphs and thereferences listed there, e.g. in Sambrook et al., Molecular Cloning,Cold Spring Habour, N.Y., 1989. The person skilled in the art will beable to identify regulation domains and binding sites of regulators bycomparing the sequence of the nucleic acid molecule of the presentinvention or the expression product thereof with the state of the art bycomputer software means which comprise algorithms for the identifying ofbinding sites and regulation domains or by introducing into a nucleicacid molecule or in a protein systematically mutations and assaying forthose mutations which will lead to an increased specifiy activity or anincreased activity per volume, in particular per cell.

It is therefore adavantageously to express in an organism a nucleic acidmolecule of the invention or a polypeptide of the invention derived froma evolutionary distantly related organism, as e.g. using a prokaryoticgene in a eukaryotic host, as in these cases the regulation mechanism ofthe host cell may not weaken the activity (cellular or specific) of thegene or its expression product

The mutation is introduced in such a way that the production of the finechemical is not adversely affected.

Less influence on the regulation of a gene or its gene product isunderstood as meaning a reduced regulation of the enzymatic activityleading to an increased specific or cellular activity of the gene or itsproduct. An increase of the enzymatic activity is understood as meaningan enzymatic activity, which is increased by at least 10%, 20%, 30%, 40%or 50%, advantageously by at least 60%, 70%, 80%, 90% or 100%,especially advantageously by at least 150%, 200%, 300% or more incomparison with the starting organism. In the event the inventivenucleic acid sequences were introduced into an organism, which did nothave the encoded protein activity, said new generated enzymatic activityshall also be embraced by the herein described invention. This leads toan increased productivity of the desired fine chemical.

Owing to the introduction of a gene or a plurality of genes conferringthe expression of the nucleic acid molecule of the invention or thepolypeptide of the invention as described below, for example the nucleicacid construct mentioned below, or e.g. encoding the protein as depictedin SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394 into an organism alone or in combination with othergenes, it is possible not only to increase the biosynthetic flux towardsthe end product, but also to increase, modify or create de novo anadvantageous, preferably novel metabolites composition in the organism,e.g. an advantageous amino acid composition comprising a higher contentof (from a viewpoint of nutrional physiology limited) amino acids, forexample essential amino acids like tryptophane, threonine, methionine orlysine.

Preferably the composition comprises further higher amounts ofmetabolites positively affecting or lower amounts of metabolitesnegatively affecting the nutrition or health of animals or humansprovided with said compositions or organisms of the invention or partsthereof. Likewise, the number or activity of further genes which arerequired for the import or export of nutrients or metabolites, includingfor example amino acids or its precursors, required for the cell'sbiosynthesis of the fine chemical may be increased so that theconcentration of necessary or relevant precursors, cofactors orintermediates within the cell(s) or within the corresponding storagecompartments is increased. Owing to the increased or novel generatedactivity of the polypeptide of the invention or owing to the increasednumber of nucleic acid sequences of the invention and/or to themodulation of further genes which are involved in the biosynthesis ofthe fine chemical, e.g. by increasing the acitivty of enzymes synthizingprecursors or by destroying the activity of one or more genes which areinvolved in the breakdown of the fine chemical, it is thereby possibleto increase the yield, production and/or production efficiency of thefine chemical in the host organism, such as plants or microorganims.

Accordingly, in one embodiment, the process according to the inventionrelates to a process, which comprises:

-   a) providing a non-human organism, preferably a microorganism,    non-human animal, plant or part, cell or tissue thereof;-   b) increasing the biological activity represented by a protein as    depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,    26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66,    68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,    102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,    128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,    154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,    180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,    206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,    232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,    258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,    284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308,    310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334,    336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,    362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386,    388, 390, 392 or 394 or of a polypeptide being encoded by the    nucleic acid molecule of the invention and described below, i.e.    conferring an increase of the fine chemical in the organism,    preferably in the microorganism, the non-human animal, the plant or    part, cell or tissue thereof,-   c) growing the organism, preferably the microorganism, the non-human    animal, the plant or part, cell or tissue thereof under conditions    which permit the production of the fine chemical; and-   d) if desired, revovering, optionally isolating, the free and/or    bound fine chemical.

The organism, in particular the microorganism, non-human animal, theplant or animal parts, the plant or animal cell, the plant or animaltissue or the plant is advantageously grown in such a way that it is notonly possible to recover, if desired isolate the free or bound finechemical (Galili et al., Transgenic Res., 2000, 9, 2, 137-144).

After the above-described increasing (which as defined above alsoencompasses the generating of an activity in an organism, i.e. a de novoactivity), for example after the introduction and the expression of thenucleic acid molecules of the invention or described in the methods orprocesses according to the invention, the organism according to theinvention, advantageously, a microorganism, a non-human animal, a plant,plant or animal tissue or plant or animal cell, is grown andsubsequently harvested.

Suitable organisms or host organisms (transgenic organism) for thenucleic acid molecule used according to the invention and for theinventive process, the nucleic acid construct or the vector (both asdescribed below) are, in principle, all organisms which are capable ofsynthesizing the fine chemical, and which are suitable for theactivation, introduction or stimulation genes. Examples which may bementioned are plants, microorganisms such as fungi, bacteria, yeasts,alga or diatom, transgenic or obtained by site directed mutagenesis orrandom mutagenesis combined with specific selection procedures.Preferred organisms are those which are naturally capable ofsynthesizing the fine chemical in substantial amounts, like fungi,yeasts, bactria or plants. In principle, transgenic animals, for exampleCaenorhabditis elegans, are also suitable as host organisms.

In the event that the transgenic organism is a microorganism, such as aeukaryotic organism, for example a fungus, an alga, diatom or a yeast inparticular a fungus, alga, diatom or yeast selected from the familiesChaetomiaceae, Choanephoraceae, Cryptococcaceae, Cunninghamellaceae,Demetiaceae, Dipodascaceae, Moniliaceae, Mortierellaceae, Mucoraceae,Pythiaceae, Saccharomycetaceae, Saprolegniaceae,Schizosaccharomycetaceae, Sodariaceae, SporobolomycetaceaeTuberculariaceae, Adelotheciaceae, Dinophyceae, Ditrichaceae orPrasinophyceae, or a fungus selected from the families Tremellaceae,Filobasidiaceae, Christianseniaceae, Cystofilobasidiaceae, Sordariaceae,Annulatascaceae, Cephalothecaceae, Chaetomiaceae, Coniochaetaceae,Lasiosphaeriaceae, Pleurotremataceae, Elaphomycetaceae, Trichocomaceae,Mucoraceae, Schizophyllaceae, Onygenaceae, Suillaceae, Hypocreaceae,Ustilaginaceae, Trichocomaceae, Phyllachoraceae, Erysiphaceae or aprokaryotic organism, for example a bacterium or blue alga, inparticular a bacterium from the families Actinomycetaceae, Bacillaceae,Brevibacteriaceae, Corynebacteriaceae, Cyanophyceae, Enterobacteriacae,Gordoniaceae, Nocardiaceae, Micrococcaceae, Mycobacteriaceae,Pseudomonaceae, Rhizobiaceae or Streptomycetaceae, this microorganism isgrown on a solid or in a liquid medium which is known to the skilledworker and suits the organism. After the growing phase, the organismscan be harvested.

The microorganisms or the recovered, and if desired isolated, finechemical can then be processed further directly into foodstuffs oranimal feeds or for other applications, for example according to thedisclosures made in EP-B-0 533 039 or EP-A-0 615 693, which areexpressly incorporated herein by reference. The fermentation broth orfermentation products can be purified in the customary manner byextraction and precipitation or via ion exchangers and other methodsknown to the person skilled in the art and described herein below.Products of these different work-up procedures are fine chemical e.g.amino acids or amino acid compositions which still comprise fermentationbroth and cell components in different amounts, advantageously in therange of from 0 to 99% by weight, preferably below 80% by weight,especially preferably between below 50%, 40%, 30%, 20%, 10% or 5% byweight.

Preferred microorganisms are selected from the group consisting ofChaetomiaceae such as the genera Chaetomium e.g. the speciesChaetomidium fimeti; Choanephoraceae such as the genera Blakeslea,Choanephora e.g. the species Blakeslea trispora, Choanephoracucurbitarum or Choanephora infundibulifera var. cucurbitarum;Cryptococcaceae such as the genera Candida, Crytococcus, Rhodotorula,Torulopsis e.g. the species Candida albicans, Candida albomarginata,Candida antarctica, Candida bacarum, Candida bogoriensis, Candidaboidinii, Candida bovina, Candida brumptii, Candida cacaoi, Candidacariosilignicola, Candida catenulata, Candida chalmersii, Candidaciferrii, Candida cylindracea, Candida edax, Candida ernobii, Candidafamata, Candida freyschussii, Candida friedrichii, Candida glabrata,Candida guilliermondii, Candida haemulonii, Candida humicola, Candidainconspicua, Candida ingens, Candida intermedia, Candida kefyr, Candidakrusei, Candida lactiscondensi, Candida lambica, Candida lipolytica,Candida lusitaniae, Candida macedoniensis, Candida magnoliae, Candidamembranaefaciens, Candida mesenterica, Candida multigemmis, Candidamycoderma, Candida nemodendra, Candida nitratophila, Candidanorvegensis, Candida norvegica, Candida parapsilosis, Candidapelliculosa, Candida peltata, Candida pini, Candida pseudotropicalis,Candida pulcherrima, Candida punicea, Candida pustula, Candida ravautii,Candida reukaufii, Candida rugosa, Candida sake, Candida silvicola,Candida solani, Candida sp., Candida spandovensis, Candida succiphila,Candida tropicalis, Candida utilis, Candida valida, Candida versatilis,Candida vini, Candida zeylanoides, Cryptococcus albidus, Cryptococcuscurvatus, Cryptococcus flavus, Cryptococcus humicola, Cryptococcushungaricus, Cryptococcus kuetzingii, Cryptococcus laurentii,Cryptococcus macerans, Cryptococcus neoformans, Cryptococcus terreus,Cryptococcus uniguttulatus, Rhodotorula acheniorum, Rhodotorula bacarum,Rhodotorula bogoriensis, Rhodotorula flava, Rhodotorula glutinis,Rhodotorula macerans, Rhodotorula minuta, Rhodotorula mucilaginosa,Rhodotorula pilimanae, Rhodotorula pustula, Rhodotorula rubra,Rhodotorula tokyoensis, Torulopsis colliculosa, Torulopsis dattila orTorulopsis neoformans; such as the genera Cunninghamella e.g. thespecies Cunninghamella blakesleeana, Cunninghamella echinulata,Cunninghamella echinulata var. elegans, Cunninghamella elegans orCunninghamella homothallica; Demetiaceae such as the genera Alternaria,Bipolaris, Cercospora, Chalara, Cladosporium, Curvularia, Exophilia,Helicosporium, Helminthosporium, Orbimyces, Philalophora, Pithomyces,Spilocaea, Thielaviopsis, Wangiella e.g. the species Curvularia affinis,Curvularia clavata, Curvularia fallax, Curvularia inaequalis, Curvulariaindica, Curvularia lunata, Curvularia pallescens, Curvularia verruculosaor Helminothosporium sp.; Moniliaceae such as the genera Arthrobotrys,Aspergillus, Epidermophyton, Geotrichum, Gliocladium, Histoplasma,Microsporum, Monilia, Oedocephalum, Oidium, Penicillium, Trichoderma,Trichophyton, Thrichoteclum, Verticillium e.g. the species Aspergillusaculeatus, Aspergillus albus, Aspergillus alliaceus, Aspergillusasperescens, Aspergillus awamori, Aspergillus candidus, Aspergilluscarbonarius, Aspergillus carneus, Aspergillus chevalieri, Aspergilluschevalieri var. intermedius, Aspergillus clavatus, Aspergillus ficuum,Aspergillus flavipes, Aspergillus flavus, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus giganteus, Aspergillus humicola,Aspergillus intermedius, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus niveus, Aspergillus ochraceus,Aspergillus oryzae, Aspergillus ostianus, Aspergillus parasiticus,Aspergillus parasiticus var. globosus, Aspergillus penicillioides,Aspergillus phoenicis, Aspergillus rugulosus, Aspergillus sclerotiorum,Aspergillus sojae var. gymnosardae, Aspergillus sydowi, Aspergillustamarai, Aspergillus terreus, Aspergillus terricola, Aspergillustoxicarius, Aspergillus unguis, Aspergillus ustus, Aspergillusversicolor, Aspergillus vitricolae, Aspergillus wentii, •Penicilliumadametzi, •Penicillium albicans, Penicillium arabicum, Penicilliumarenicola, Penicillium argillaceum, Penicillium arvense, Penicilliumasperosporum, •Penicillium aurantiogriseum, •Penicillium avellaneum,•Penicillium baarnense, •Penicillium bacillisporum, •Penicilliumbrasilianum, •Penicillium brevicompactum, •Penicillium camemberti,•Penicillium canadense, •Penicillium canescens, •Penicillium caperatum,•Penicillium capsulatum, •Penicillium caseicolum, •Penicilliumchrysogenum, •Penicillium citreonigrum, •Penicillium citrinum,•Penicillium claviforme, •Penicillium commune, •Penicilliumcorylophilum, •Penicillium corymbiferum, •Penicillium crustosum,•Penicillium cyclopium, •Penicillium daleae, •Penicillium decumbens,•Penicillium dierckxii, •Penicillium digitatum, •Penicillium digitatumvar. latum, •Penicillium divaricatum, •Penicillium diversum,•Penicillium duclauxii, •Penicillium echinosporum, •Penicilliumexpansum, •Penicillium fellutanum, •Penicillium frequentans,•Penicillium funiculosum, •Penicillium glabrum, •Penicillium gladioli,•Penicillium griseofulvum, •Penicillium hirsutum, •Penicilliumhispanicum, •Penicillium islandicum, •Penicillium italicum, •Penicilliumitalicum var. avellaneum, •Penicillium janczewskii, •Penicilliumjanthinellum, •Penicillium japonicum, •Penicillium lavendulum,•Penicillium lilacinum, •Penicillium lividum, •Penicillium martensii,Penicillium megasporum, •Penicillium miczynskii, •Penicilliumnalgiovense, •Penicillium nigricans, Penicillium notatum, •Penicilliumochrochloron, •Penicillium odoratum, •Penicillium oxalicum, •Penicilliumparaherquei, •Penicillium patulum, •Penicillium pinophilum, •Penicilliumpiscarium, •Penicillium pseudostromaticum, •Penicillium puberulum,•Penicillium purpurogenum, •Penicillium raciborskii, •Penicilliumroqueforti, •Penicillium rotundum, •Penicillium rubrum, •Penicilliumsacculum, •Penicillium simplicissimum, Penicillium sp., Penicilliumspinulosum, Penicillium steckii, Penicillium stoloniferum, Penicilliumstriatisporum, Penicillium striatum, Penicillium tardum, Penicilliumthomii, Penicillium turbatum, Penicillium variabile, Penicilliumvermiculatum, Penicillium vermoesenii, Penicillium verrucosum,Penicillium verrucosum var. corymbiferum, Penicillium verrucosum var.cyclopium, Penicillium verruculosum, Penicillium vinaceum, Penicilliumviolaceum, Penicillium viridicatum, Penicillium vulpinum, Trichodermahamatum, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma polysporum, Trichoderma reesei, Trichodermavirens or Trichoderma viride; Mortierellaceae such as the generaMortierella e.g. the species Mortierella isabellina, Mortierellapolycephala, Mortierella ramanniana, Mortierella vinacea or Mortierellazonata; Mucoraceae such as the genera Actinomucor, Mucor, Phycomyces,Rhizopus, Zygorhynchus e.g. the species Mucor amphibiorum, Mucorcircinelloides f. circinelloides, Mucor circinelloides var.griseocyanus, Mucor flavus, Mucor fuscus, Mucor griseocyanus, Mucorheterosporus, Mucor hiemalis, Mucor hiemalis f. hiemalis, Mucorinaequisporus, Mucor indicus, Mucor javanicus, Mucor mucedo, Mucormucilagineus, Mucor piriformis, Mucor plasmaticus, Mucor plumbeus, Mucorracemosus, Mucor racemosus f. racemosus, Mucor racemosus f.sphaerosporus, Mucor rouxianus, Mucor rouxii, Mucor sinensis, Mucor sp.,Mucor spinosus, Mucor tuberculisporus, Mucor varlisporus, Mucorvariosporus, Mucor wosnessenskii, Phycomyces blakesleeanus, Rhizopusachlamydosporus, Rhizopus arrhizus, Rhizopus chinensis, Rhizopusdelemar, Rhizopus formosaensis, Rhizopus japonicus, Rhizopus javanicus,Rhizopus microsporus, Rhizopus microsporus var. chinensis, Rhizopusmicrosporus var. oligosporus, Rhizopus microsporus var. rhizopodiformis,Rhizopus nigricans, Rhizopus niveus, Rhizopus oligosporus, Rhizopusoryzae, Rhizopus pygmaeus, Rhizopus rhizopodiformis, Rhizopussemarangensis, Rhizopus sontii, Rhizopus stolonifer, Rhizopus thermosus,Rhizopus tonkinensis, Rhizopus tritici or Rhizopus usamii; Pythiaceaesuch as the genera Phytium, Phytophthora e.g. the species Pythiumdebaryanum, Pythium intermedium, Pythium irregulare, Pythiummegalacanthum, Pythium paroecandrum, Pythium sylvaticum, Pythiumultimum, Phytophthora cactorum, Phytophthora cinnamomi, Phytophthoracitricola, Phytophthora citrophthora, Phytophthora cryptogea,Phytophthora drechsleri, Phytophthora erythroseptica, Phytophthoralateralis, Phytophthora megasperma, Phytophthora nicotianae,Phytophthora nicotianae var. parasitica, Phytophthora palmivora,Phytophthora parasitica or Phytophthora syringae; Saccharomycetaceaesuch as the genera Hansenula, Pichia, Saccharomyces, Saccharomycodes,Yarrowia e.g. the species Hansenula anomala, Hansenula californica,Hansenula canadensis, Hansenula capsulata, Hansenula ciferrii, Hansenulaglucozyma, Hansenula henricii, Hansenula holstii, Hansenula minuta,Hansenula nonfermentans, Hansenula philodendri, Hansenula polymorpha,Hansenula saturnus, Hansenula subpelliculosa, Hansenula wickerhamii,Hansenula wingei, Pichia alcoholophila, Pichia angusta, Pichia anomala,Pichia bispora, Pichia burtonii, Pichia canadensis, Pichia capsulata,Pichia carsonii, Pichia cellobiosa, Pichia ciferrii, Pichia farinosa,Pichia fermentans, Pichia finlandica, Pichia glucozyma, Pichiaguilliermondii, Pichia haplophila, Pichia henricii, Pichia holstii,Pichia jadinii, Pichia lindnerii, Pichia membranaefaciens, Pichiamethanolica, Pichia minuta var. minuta, Pichia minuta var.nonfermentans, Pichia norvegensis, Pichia ohmeri, Pichia pastoris,Pichia philodendri, Pichia pini, Pichia polymorpha, Pichia quercuum,Pichia rhodanensis, Pichia sargentensis, Pichia stipitis, Pichiastrasburgensis, Pichia subpelliculosa, Pichia toletana, Pichiatrehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceti,Saccharomyces bailii, Saccharomyces bayanus, Saccharomyces bisporus,Saccharomyces capensis, Saccharomyces carisbergensis, Saccharomycescerevisiae, Saccharomyces cerevisiae var. ellipsoideus, Saccharomyceschevalieri, Saccharomyces delbrueckii, Saccharomyces diastaticus,Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomycesellipsoideus, Saccharomyces fermentati, Saccharomyces florentinus,Saccharomyces fragilis, Saccharomyces heterogenicus, Saccharomyceshienipiensis, Saccharomyces inusitatus, Saccharomyces italicus,Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis,Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomycesmontanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomycesparadoxus, Saccharomyces pastorianus, Saccharomyces pretoriensis,Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum,Saccharomycodes ludwigii or Yarrowia lipolytica; Saprolegniaceae such asthe genera Saprolegnia e.g. the species Saprolegnia ferax;Schizosaccharomycetaceae such as the genera Schizosaccharomyces e.g. thespecies Schizosaccharomyces japonicus var. japonicus,Schizosaccharomyces japonicus var. versatilis, Schizosaccharomycesmalidevorans, Schizosaccharomyces octosporus, Schizosaccharomyces pombevar. malidevorans or Schizosaccharomyces pombe var. pombe; Sodariaceaesuch as the genera Neurospora, Sordaria e.g. the species Neurosporaafricana, Neurospora crassa, Neurospora intermedia, Neurosporasitophila, Neurospora tetrasperma, Sordaria fimicola or Sordariamacrospora; Tuberculariaceae such as the genera Epicoccum, Fusarium,Myrothecium, Sphacelia, Starkeyomyces, Tubercularia e.g. the speciesFusarium acuminatum, Fusarium anthophilum, Fusarium aquaeductuum,Fusarium aquaeductuum var. medium, Fusarium avenaceum, Fusariumbuharicum, Fusarium camptoceras, Fusarium cerealis, Fusanumchlamydosporum, Fusarium ciliatum, Fusarium coccophilum, Fusariumcoeruleum, Fusarium concolor, Fusarium crookwellense, Fusarium culmorum,Fusarium dimerum, Fusarium diversisporum, Fusarium equiseti, Fusanumequiseti var. bullatum, Fusarium eumartii, Fusarium flocciferum,Fusarium fujikuroi, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium incarnatum, Fusarium inflexum, Fusariumjavanicum, Fusarium lateritium, Fusarium lateritium var. majus, Fusariumlongipes, Fusarium melanochlorum, Fusarium merismoides, Fusariummerismoides var. chlamydosporale, Fusarium moniliforme, Fusariummoniliforme var. anthophilum, Fusarium moniliforme var. subglutinans,Fusarium nivale, Fusarium nivale var. majus, Fusarium oxysporum,Fusarium oxysporumf. sp. aechmeae, Fusarium oxysporumf. sp. cepae,Fusarium oxysporumf. sp. conglutinans, Fusarium oxysporumf. sp.cucumerinum, Fusarium oxysporumf. sp. cyclaminis, Fusarium oxysporumf.sp. dianthi, Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporumf.sp. melonis, Fusarium oxysporumf. sp. passiflorae, Fusarium oxysporumf.sp. pisi, Fusarium oxysporumf. sp. tracheiphilum, Fusarium oxysporumf.sp. tuberosi, Fusarium oxysporumf. sp. tulipae, Fusarium oxysporumf. sp.vasinfectum, Fusarium pallidoroseum, Fusarium poae, Fusariumproliferatum, Fusarium proliferatum var. minus, Fusarium redolens,Fusarium redolens f. sp. dianthi, Fusarium reticulatum, Fusarium roseum,Fusarium sacchari var. elongatum, Fusarium sambucinum, Fusariumsambucinum var. coeruleum, Fusarium semitectum, Fusarium semitectum var.majus, Fusarium solani, Fusarium solanif. sp. pisi, Fusariumsporotrichioides, Fusarium sporotrichioides var. minus, Fusariumsublunatum, Fusarium succisae, Fusarium sulphureum, Fusarium tabacinum,Fusarium tricinctum, Fusarium udum, Fusarium ventricosum, Fusariumverticillioides, Fusarium xylarioides or Fusarium zonatum;Sporobolomycetaceae such as the genera Bullera, Sporobolomyces,Itersonilia e.g. the species Sporobolomyces holsaticus, Sporobolomycesodorus, Sporobolomyces puniceus, Sporobolomyces salmonicolor,Sporobolomyces singularis or Sporobolomyces tsugae; Adelotheciaceae suchas the genera e.g. the species Physcomitrella patens; Dinophyceae suchas the genera Crypthecodinium, Phaeodactylum e.g. the speciesCrypthecodinium cohnii or Phaeodactylum tricornutum; Ditrichaceae suchas the genera Ceratodon, Pleuridium, Astomiopsis, Ditrichum,Philibertiella, Ceratodon, Distichium, Skottsbergia e.g. the speciesCeratodon antarcticus, Ceratodon purpureus, Ceratodon purpureus ssp.convolutes or Ceratodon purpureus ssp. stenocarpus; Prasinophyceae suchas the genera Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis,Mantoniella, Ostreococcus e.g. the species Nephroselmis olivacea,Prasinococcus capsulatus, Scherffelia dubia, Tetraselmis chui,Tetraselmis suecica, Mantoniella squamata or Ostreococcus tauri;Actinomycetaceae such as the genera Actinomyces, Actinobaculum,Arcanobacterium, Mobiluncus e.g. the species Actinomyces bernardiae,Actinomyces bovis, Actinomyces bowdenii, Actinomyces canis, Actinomycescardiffensis, Actinomyces catuli, Actinomyces coleocanis, Actinomycesdenticolens, Actinomyces europaeus, Actinomyces funkei, Actinomycesgeorgiae, Actinomyces gerencseriae, Actinomyces hordeovulneris,Actinomyces howellii, Actinomyces humiferus, Actinomyces hyovaginalis,Actinomyces israelii Actinomyces marimammalium, Actinomyces meyeri,Actinomyces naeslundii, Actinomyces nasicola, Actinomyces neuii subsp.anitratus, Actinomyces neuii subsp. neuii, Actinomyces odontolyticus,Actinomyces oricola, Actinomyces pyogenes, Actinomyces radicidentis,Actinomyces radingae, Actinomyces slackii, Actinomyces suimastitidis,Actinomyces suis, Actinomyces turicensis, Actinomyces urogenitalis,Actinomyces vaccimaxillae, Actinomyces viscosus, Actinobaculum schaalii,Actinobaculum suis, Actinobaculum urinale, Arcanobacterium bernardiae,Arcanobacterium haemolyticum, Arcanobacterium hippocoleae,Arcanobacterium phocae, Arcanobacterium pluranimalium, Arcanobacteriumpyogenes, Mobiluncus curtisii subsp. curtisii, Mobiluncus curtisiisubsp. holmesii or Mobiluncus mulieris; Bacillaceae such as the generaAmphibacillus, Anoxybacillus, Bacillus, Exiguobacterium,Gracilibacillus, Holobacillus, Saccharococcus, Salibacillus,Virgibacillus e.g. the species Amphibacillus fermentum, Amphibacillustropicus, Amphibacillus xylanus, Anoxybacillus flavithermus,Anoxybacillus gonensis, Anoxybacillus pushchinoensis, Bacillusacidocaldarius, Bacillus acidoterrestris, Bacillus aeolius, Bacillusagaradhaerens, Bacillus agri, Bacillus alcalophilus, Bacillusalginolyticus, Bacillus alvei, Bacillus amyloliquefaciens, Bacillusamylolyticus, Bacillus aneurinilyticus, Bacillus aquimaris, Bacillusarseniciselenatis, Bacillus atrophaeus, Bacillus azotofixans, Bacillusazotoformans, Bacillus badius, Bacillus barbaricus, Bacillusbenzoevorans, Bacillus borstelensis, Bacillus brevis, Bacilluscarboniphilus, Bacillus centrosporus, Bacillus cereus, Bacilluschitinolyticus, Bacillus chondroitinus, Bacillus choshinensis, Bacilluscirculans, Bacillus clarkii, Bacillus clausii, Bacillus coagulans,Bacillus cohnii, Bacillus curdlanolyticus, Bacillus cycloheptanicus,Bacillus decolorationis, Bacillus dipsosauri, Bacillus edaphicus,Bacillus ehimensis, Bacillus endophyticus, Bacillus fastidiosus,Bacillus firmus, Bacillus flexus, Bacillus formosus, Bacillus fumarioli,Bacillus funiculus, Bacillus fusiformis, Bacillus sphaericus subsp.fusiformis, Bacillus galactophilus, Bacillus globisporus, Bacillusglobisporus subsp. marinus, Bacillus glucanolyticus, Bacillus gordonae,Bacillus halmapalus, Bacillus haloalkaliphilus, Bacillushalodenitrificans, Bacillus halodurans, Bacillus halophilus, Bacillushorikoshii, Bacillus horti, Bacillus infernos, Bacillus insolitus,Bacillus jeotgali, Bacillus kaustophilus, Bacillus kobensis, Bacilluskrulwichiae, Bacillus laevolacticus, Bacillus larvae, Bacilluslaterosporus, Bacillus lautus, Bacillus lentimorbus, Bacillus lentus,Bacillus licheniformis, Bacillus luciferensis, Bacillus macerans,Bacillus macquarensis, Bacillus marinus, Bacillus marisflavi, Bacillusmarismortui, Bacillus megaterium, Bacillus methanolicus, Bacillusmigulanus, Bacillus mojavensis, Bacillus mucilaginosus, Bacillusmycoides, Bacillus naganoensis, Bacillus nealsonii, Bacillus neidei,Bacillus niacini, Bacillus okuhidensis, Bacillus oleronius, Bacilluspabuli, Bacillus pallidus, Bacillus pantothenticus, Bacillus parabrevis,Bacillus pasteurii, Bacillus peoriae, Bacillus polymyxa, Bacilluspopilliae, Bacillus pseudalcaliphilus, Bacillus pseudofirmus, Bacilluspseudomycoides, Bacillus psychrodurans, Bacillus psychrophilus, Bacilluspsychrosaccharolyticus, Bacillus psychrotolerans, Bacillus pulvifaciens,Bacillus pumilus, Bacillus pycnus, Bacillus reuszeri, Bacillussalexigens, Bacillus schlegelii, Bacillus selenitireducens, Bacillussilvestris, Bacillus simplex, Bacillus siralis, Bacillus smithii,Bacillus sonorensis, Bacillus sphaericus, Bacillus sporothermodurans,Bacillus stearothermophilus, Bacillus subterraneus, Bacillus subtilissubsp. spizizenii, Bacillus subtilis subsp. subtilis, Bacillusthermantarcticus, Bacillus thermoaerophilus, Bacillus thermoamylovorans,Bacillus thermoantarcticus, Bacillus thermocatenulatus, Bacillusthermocloacae, Bacillus thermodenitrificans, Bacillusthermoglucosidasius, Bacillus thermoleovorans, Bacillus thermoruber,Bacillus thermosphaericus, Bacillus thiaminolyticus, Bacillusthuringiensis, Bacillus tusciae, Bacillus validus, Bacillusvallismortis, Bacillus vedderi, Bacillus vulcani, Bacillusweihenstephanensis, Exiguobacterium acetylicum, Exiguobacteriumantarcticum, Exiguobacterium aurantiacum, Exiguobacterium undae,Gracilibacillus dipsosauri, Gracilibacillus halotolerans, Halobacillushalophilus, Halobacillus karajensis, Halobacillus litoralis,Halobacillus salinus, Halobacillus trueperi, Saccharococcuscaldoxylosilyticus, Saccharococcus thermophilus, Salibacillusmarismortui, Salibacillus salexigens, Virgibacillus carmonensis,Virgibacillus marismortui, Virgibacillus necropolis, Virgibacilluspantothenticus, Virgibacillus picturae, Virgibacillus proomii orVirgibacillus salexigens, Brevibacteriaceae such as the generaBrevibacterium e.g. the species Brevibacterium acetylicum,Brevibacterium albidum, Brevibacterium ammoniagenes, Brevibacteriumavium, Brevibacterium casei, Brevibacterium citreum, Brevibacteriumdivaricatum, Brevibacterium epidermidis, Brevibacterium fermentans,Brevibacterium frigoritolerans, Brevibacterium halotolerans,Brevibacterium imperiale, Brevibacterium incertum, Brevibacteriumiodinum, Brevibacterium linens, Brevibacterium liquefaciens,Brevibacterium lutescens, Brevibacterium luteum, Brevibacterium lyticum,Brevibacterium mcbrellneri, Brevibacterium otitidis, Brevibacteriumoxydans, Brevibacterium paucivorans, Brevibacterium protophormiae,Brevibacterium pusillum, Brevibacterium saperdae, Brevibacteriumstationis, Brevibacterium testaceum or Brevibacterium vitaeruminis;Corynebacteriaceae such as the genera Corynebacterium e.g. the speciesCorynebacterium accolens, Corynebacterium afermentans subsp.afermentans, Corynebacterium afermentans subsp. lipophilum,Corynebacterium ammoniagenes, Corynebacterium amycolatum,Corynebacterium appendicis, Corynebacterium aquilae, Corynebacteriumargentoratense, Corynebacterium atypicum, Corynebacterium aurimucosum,Corynebacterium auris, Corynebacterium auriscanis, Corynebacteriumbetae, Corynebacterium beticola, Corynebacterium bovis, Corynebacteriumcallunae, Corynebacterium camporealensis, Corynebacterium capitovis,Corynebacterium casei, Corynebacterium confusum, Corynebacteriumcoyleae, Corynebacterium cystitidis, Corynebacterium durum,Corynebacterium efficiens, Corynebacterium equi, Corynebacteriumfalsenii, Corynebacterium fascians, Corynebacterium felinum,Corynebacterium flaccumfaciens, Corynebacterium flavescens,Corynebacterium freneyi, Corynebacterium glaucum, Corynebacteriumglucuronolyticum, Corynebacterium glutamicum, Corynebacterium hoagii,Corynebacterium ilicis, Corynebacterium imitans, Corynebacteriuminsidiosum, Corynebacterium iranicum, Corynebacterium jeikeium,Corynebacterium kroppenstedtii, Corynebacterium kutscheri,Corynebacterium lilium, Corynebacterium lipophiloflavum, Corynebacteriummacginleyi, Corynebacterium mastitidis, Corynebacterium matruchotii,Corynebacterium michiganense, Corynebacterium michiganense subsp.tessellarius, Corynebacterium minutissimum, Corynebacteriummooreparkense, Corynebacterium mucifaciens, Corynebacterium mycetoides,Corynebacterium nebraskense, Corynebacterium oortii, Corynebacteriumpaurometabolum, Corynebacterium phocae, Corynebacterium pilosum,Corynebacterium poinsettiae, Corynebacterium propinquum, Corynebacteriumpseudodiphtheriticum, Corynebacterium pseudotuberculosis,Corynebacterium pyogenes, Corynebacterium rathayi, Corynebacteriumrenale, Corynebacterium riegelii, Corynebacterium seminale,Corynebacterium sepedonicum, Corynebacterium simulans, Corynebacteriumsingulare, Corynebacterium sphenisci, Corynebacterium spheniscorum,Corynebacterium striatum, Corynebacterium suicordis, Corynebacteriumsundsvallense, Corynebacterium terpenotabidum, Corynebacteriumtestudinoris, Corynebacterium thomssenii, Corynebacterium tritici,Corynebacterium ulcerans, Corynebacterium urealyticum, Corynebacteriumvariabile, Corynebacterium vitaeruminis or Corynebacterium xerosis;Enterobacteriacae such as the genera Alterococcus, Arsenophonus,Brenneria, Buchnera, Budvicia, Buttiauxella, Calymmatobacterium,Cedecea, Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia,Ewingelia, Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella,Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium,Photorhabdus, Plesiomonas, Pragia, Proteus, Providencia, Rahnella,Saccharobacter, Salmonella, Shigella, Serratia, Sodalis, Tatumella,Trabulsiella, Wigglesworthia, Xenorhabdus,Yersinia and Yokenella e.g.the species Arsenophonus nasoniae, Brenneria alni, Brennerianigrifluens, Brenneria quercina, Brenneria rubrifaciens, Brenneriasalicis, Budvicia aquatica, Buttiauxella agrestis, Buttiauxellabrennerae, Buttiauxella ferragutiae, Buttiauxella gaviniae, Buttiauxellaizardii, Buttiauxella noackiae, Buttiauxella warmboldiae, Cedeceadavisae, Cedecea lapagei, Cedecea neteri, Citrobacter amalonaticus,Citrobacter diversus, Citrobacter freundii, Citrobacter genomospecies,Citrobacter gilleni, Citrobacter intermedium, Citrobacter koseri,Citrobacter murliniae, Citrobacter sp., Edwardsiella hoshinae,Edwardsiella ictaluri, Edwardsiella tarda, Erwinia alni, Erwiniaamylovora, Erwinia ananatis, Erwinia aphidicola, Erwinia billingiae,Erwinia cacticida, Erwinia cancerogena, Erwinia carnegieana, Erwiniacarotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum,Erwinia carotovora subsp. odorifera, Erwinia carotovora subsp. wasabiae,Erwinia chrysanthemi, Erwinia cypripedii, Erwinia dissolvens, Erwiniaherbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens,Erwinia nimipressuralis, Erwinia persicina, Erwinia psidii, Erwiniapyrifoliae, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens,Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwiniauredovora, Escherichia adecarboxylata, Escherichia anindolica,Escherichia aurescens, Escherichia blattae, Escherichia coli,Escherichia coli var. communior, Escherichia coli-mutabile, Escherichiafergusonii, Escherichia hermannii, Escherichia sp., Escherichiavulneris, Ewingella americana, Hafnia alvei, Klebsiella aerogenes,Klebsiella edwardsii subsp. atlantae, Klebsiella ornithinolytica,Klebsiella oxytoca, Klebsiella planticola, Klebsiella pneumoniae,Klebsiella pneumoniae subsp. pneumoniae, Klebsiella sp., Klebsiellaterrigena, Klebsiella trevisanii, Kluyvera ascorbata, Kluyveracitrophila, Kluyvera cochleae, Kluyvera cryocrescens, Kluyverageorgiana, Kluyvera noncitrophila, Kluyvera sp., Leclerciaadecarboxylata, Leminorella grimontii, Leminorella richardii,Moellerella wisconsensis, Morganella morganii, Morganella morganiisubsp. morganii, Morganella morganii subsp. sibonli, Obesumbateriumproteus, Pantoea agglomerans, Pantoea ananatis, Pantoea citrea, Pantoeadispersa, Pantoea punctata, Pantoea stewartii subsp. stewartii, Pantoeaterrea, Pectobacterium atrosepticum, Pectobacterium carotovorum subsp.atrosepticum, Pectobacterium carotovorum subsp. carotovorum,Pectobacterium chrysanthemi, Pectobacterium cypripedii, Photorhabdusasymbiotica, Photorhabdus luminescens, Photorhabdus luminescens subsp.akhurstii, Photorhabdus luminescens subsp. laumondii, Photorhabdusluminescens subsp. luminescens, Photorhabdus sp., Photorhabdustemperata, Plesiomonas shigelloides, Pragia fontium, Proteus hauseri,Proteus ichthyosmius, Proteus inconstans, Proteus mirabilis, Proteusmorganii, Proteus myxofaciens, Proteus penneri, Proteus rettgeri,Proteus shigelloides, Proteus vulgaris, Providencia alcalifaciens,Providencia friedericiana, Providencia heimbachae, Providencia rettgeri,Providencia rustigianii, Providencia stuartii, Rahnella aquatilis,Salmonella abony, Salmonella arizonae, Salmonella bongori, Salmonellacholeraesuis subsp. arizonae, Salmonella choleraesuis subsp. bongori,Salmonella choleraesuis subsp. cholereasuis, Salmonella choleraesuissubsp. diarizonae, Salmonella choleraesuis subsp. houtenae, Salmonellacholeraesuis subsp. indica, Salmonella choleraesuis subsp. salamae,Salmonella daressalaam, Salmonella enterica subsp. houtenae, Salmonellaenterica subsp. salamae, Salmonella enteritidis, Salmonella gallinarum,Salmonella heidelberg, Salmonella panama, Salmonella senftenberg,Salmonella typhimurium, Serratia entomophila, Serratia ficaria, Serratiafonticola, Serratia grimesii, Serratia liquefaciens, Serratiamarcescens, Serratia marcescens subsp. marcescens, Serratia madinorubra,Serratia odorifera, Serratia plymouthensis, Serratia plymuthica,Serratia proteamaculans, Serratia proteamaculans subsp. quinovora,Serratia quinivorans, Serratia rubidaea, Shigella boydii, Shigellaflexneri, Shigella paradysenteriae, Shigella sonnei, Tatumella ptyseos,Xenorhabdus beddingli, Xenorhabdus bovienii, Xenorhabdus luminescens,Xenorhabdus nematophila, Xenorhabdus nematophila subsp. beddingii,Xenorhabdus nematophila subsp. bovienii, Xenorhabdus nematophila subsp.poinardi or Xenorhabdus poinarii; Gordoniaceae such as the generaGordonia, Skermania e.g. the species Gordonia aichiensis, Gordoniaalkanivorans, Gordonia amarae, Gordonia amicalis, Gordonia bronchialis,Gordonia desulfuricans, Gordonia hirsuta, Gordonia hydrophobica,Gordonia namibiensis, Gordonia nitida, Gordonia paraffinivorans,Gordonia polyisoprenivorans, Gordonia rhizosphera, Gordoniarubripertincta, Gordonia sihwensis, Gordonia sinesedis, Gordonia sputi,Gordonia terrae or Gordonia westfalica; Micrococcaceae such as thegenera Micrococcus, Arthrobacter, Kocuria, Nesterenkonia, Renibacterium,Rothia, Stomatococcus e.g. the species Micrococcus agilis, Micrococcusantarcticus, Micrococcus halobius, Micrococcus kristinae, Micrococcusluteus, Micrococcus lylae, Micrococcus nishinomiyaensis, Micrococcusroseus, Micrococcus sedentarius, Micrococcus varians, Arthrobacteragilis, Arthrobacter albus, Arthrobacter atrocyaneus, Arthrobacteraurescens, Arthrobacter chlorophenolicus, Arthrobacter citreus,Arthrobacter creatinolyticus, Arthrobacter crystallopoietes,Arthrobacter cumminsii, Arthrobacter duodecadis, Arthrobacterflavescens, Arthrobacter flavus, Arthrobacter gandavensis, Arthrobacterglobiformis, Arthrobacter histidinolovorans, Arthrobacter ilicis,Arthrobacter koreensis, Arthrobacter luteolus, Arthrobactermethylotrophus, Arthrobacter mysorens, Arthrobacter nasiphocae,Arthrobacter nicotianae, Arthrobacter nicotinovorans, Arthrobacteroxydans, Arthrobacter pascens, Arthrobacter picolinophilus, Arthrobacterpolychromogenes, Arthrobacter protophormiae, Arthrobacterpsychrolactophilus, Arthrobacter radiotolerans, Arthrobacter ramosus,Arthrobacter rhombi, Arthrobacter roseus, Arthrobacter siderocapsulatus,Arthrobacter simplex, Arthrobacter sulfonivorans, Arthrobactersulfureus, Arthrobacter terregens, Arthrobacter tumescens, Arthrobacteruratoxydans, Arthrobacter ureafaciens, Arthrobacter variabilis,Arthrobacter viscosus, Arthrobacter woluwensis, Kocuria erythromyxa,Kocuria kristinae, Kocuria palustris, Kocuria polaris, Kocuriarhizophila, Kocuria rosea, Kocuria varians, Nesterenkonia halobia,Nesterenkonia lacusekhoensis, Renibacterium salmoninarum, Rothia amarae,Rothia dentocariosa, Rothia mucilaginosa, Rothia nasimurium orStomatococcus mucilaginosus; Mycobacteriaceae such as the generaMycobacterium e.g. the species Mycobacterium afficanum, Mycobactenumagri, Mycobacterium aichiense, Mycobacterium alvei, Mycobacteriumasiaticum, Mycobacterium aurum, Mycobacterium austroafricanum,Mycobacterium bohemicum, Mycobacterium botniense, Mycobacterium brumae,Mycobacterium chelonae subsp. abscessus, Mycobacterium chitae,Mycobacterium chlorophenolicum, Mycobacterium chubuense, Mycobacteriumconfluentis, Mycobacterium cookii, Mycobacterium diernhoferi,Mycobacterium doricum, Mycobacterium duvalii, Mycobacterium fallax,Mycobacterium farcinogenes, Mycobacterium flavescens, Mycobacteriumfrederiksbergense, Mycobacterium gadium, Mycobacterium gilvum,Mycobacterium gordonae, Mycobacterium hassiacum, Mycobacteriumhiberniae, Mycobacterium hodleri, Mycobacterium holsaticum,Mycobacterium komossense, Mycobacterium lacus, Mycobacteriummadagascariense, Mycobacterium mageritense, Mycobacterium montefiorense,Mycobacterium moriokaense, Mycobacterium murale, Mycobacterium neoaurum,Mycobacterium nonchromogenicum, Mycobacterium obuense, Mycobacteriumpalustre, Mycobacterium parafortuitum, Mycobacterium peregrinum,Mycobacterium phlei, Mycobacterium pinnipedii, Mycobacterium poriferae,Mycobacterium pulveris, Mycobacterium rhodesiae, Mycobacterium shottsii,Mycobacterium sphagni, Mycobacterium terrae, Mycobacteriumthermoresistibile, Mycobacterium tokaiense, Mycobacterium triviale,Mycobactenium tusciae or Mycobacterium vanbaalenii; Nocardiaceae such asthe genera Nocardia, Rhodococcus e.g. the species Nocardia abscessus,Nocardia africana, Nocardia amarae, Nocardia asteroides, Nocardiaautotrophica, Nocardia beijingensis, Nocardia brasiliensis, Nocardiabrevicatena, Nocardia caishijiensis, Nocardia calcarea, Nocardia carnea,Nocardia cellulans, Nocardia cerradoensis, Nocardia coeliaca, Nocardiacorynebacterioides, Nocardia crassostreae, Nocardia cummidelens,Nocardia cyriacigeorgica, Nocardia farcinica, Nocardia flavorosea,Nocardia fluminea, Nocardia globerula, Nocardia hydrocarbonoxydans,Nocardia ignorata, Nocardia mediterranei, Nocardia nova, Nocardiaorientalis, Nocardia otitidiscaviarum, Nocardia otitidiscaviarum,Nocardia paucivorans, Nocardia petroleophila, Nocardia pinensis,Nocardia pseudobrasiliensis, Nocardia pseudovaccinii, Nocardia puris,Nocardia restricta, Nocardia rugosa, Nocardia salmonicida, Nocardiasaturnea, Nocardia seriolae, Nocardia soli, Nocardia sulphurea, Nocardiatransvalensis, Nocardia uniformis, Nocardia vaccinii, Nocardia veteranaor Nocardia vinacea; Pseudomonaceae such as the genera Azomonas,Azotobacter, Cellvibrio, Chryseomonas, Flaviomonas, Lampropedia,Mesophilobacter, Morococcus, Oligella, Pseudomonas, Rhizobacter,Rugamonas, Serpens, Thermoleophilum, Xylophilus e.g. the speciesAzomonas agilis, Azomonas insignis, Azomonas macrocytogenes, Azotobacteragilis, Azotobacter agilis subsp. armeniae, Azotobacter armeniacus,Azotobacter beijerinckii, Azotobacter chroococcum, Azotobacter indicum,Azotobacter macrocytogenes, Azotobacter miscellum, Azotobacter nigricanssubsp. nigricans, Azotobacter paspali, Azotobacter salinestris,Azotobacter sp., Azotobacter vinelandii, Flavimonas oryzihabitans,Mesophilobacter marinus, Oligella urethralis, Pseudomonas acidovorans,Pseudomonas aeruginosa, Pseudomonas agarici, Pseudomonas alcaligenes,Pseudomonas aminovorans, Pseudomonas amygdali, Pseudomonas andropogonis,Pseudomonas anguilliseptica, Pseudomonas antarctica, Pseudomonasantimicrobica, Pseudomonas antimycetica, Pseudomonas aptata, Pseudomonasarvilla, Pseudomonas asplenii, Pseudomonas atlantica, Pseudomonasatrofaciens, Pseudomonas aureofaciens, Pseudomonas avellanae,Pseudomonas azelaica, Pseudomonas azotocolligans, Pseudomonas balearica,Pseudomonas barkeri, Pseudomonas bathycetes, Pseudomonas beijeinckii,Pseudomonas brassicacearum, Pseudomonas brenneri, Pseudomonasbutanovora, Pseudomonas carboxydoflava, Pseudomonas carboxydohydrogena,Pseudomonas carboxydovorans, Pseudomonas carrageenovora, Pseudomonascaryophylli, Pseudomonas cepacia, Pseudomonas chloritidismutans,Pseudomonas chlororaphis, Pseudomonas cichorii, Pseudomonascitronellolis, Pseudomonas cocovenenans, Pseudomonas compransoris,Pseudomonas congelans, Pseudomonas coronafaciens, Pseudomonas corrugata,Pseudomonas dacunhae, Pseudomonas delafieldii, Pseudomonas delphinii,Pseudomonas denitrificans, Pseudomonas desmolytica, Pseudomonasdiminuta, Pseudomonas doudoroffii, Pseudomonas echinoides, Pseudomonaselongata, Pseudomonas extorquens, Pseudomonas extremorientalis,Pseudomonas facilis, Pseudomonas ficuserectae, Pseudomonas flava,Pseudomonas flavescens, Pseudomonas fluorescens, Pseudomonas fragi,Pseudomonas frederiksbergensis, Pseudomonas fulgida, Pseudomonasfuscovaginae, Pseudomonas gazotropha, Pseudomonas gladioli, Pseudomonasglathei, Pseudomonas glumae, Pseudomonas graminis, Pseudomonashalophila, Pseudomonas helianthi, Pseudomonas huttiensis, Pseudomonashydrogenothermophila, Pseudomonas hydrogenovora, Pseudomonas indica,Pseudomonas indigofera, Pseudomonas iodinum, Pseudomonas kilonensis,Pseudomonas lachrymans, Pseudomonas lapsa, Pseudomonas lemoignei,Pseudomonas lemonnieri, Pseudomonas lundensis, Pseudomonas luteola,Pseudomonas maltophilia, Pseudomonas marginalis, Pseudomonas marginata,Pseudomonas marina, Pseudomonas meliae, Pseudomonas mendocina,Pseudomonas mesophilica, Pseudomonas mixta, Pseudomonas monteilii,Pseudomonas morsprunorum, Pseudomonas multivorans, Pseudomonasnatriegens, Pseudomonas nautica, Pseudomonas nitroreducens, Pseudomonasoleovorans, Pseudomonas oryzihabitans, Pseudomonas ovalis, Pseudomonasoxalaticus, Pseudomonas palleronii, Pseudomonas paucimobilis,Pseudomonas phaseolicola, Pseudomonas phenazinium, Pseudomonaspickettii, Pseudomonas pisi, Pseudomonas plantarii, Pseudomonasplecoglossicida, Pseudomonas poae, Pseudomonas primulae, Pseudomonasproteolytica, Pseudomonas pseudoalcaligenes, Pseudomonaspseudoalcaligenes subsp. konjaci, Pseudomonas pseudoalcaligenes subsp.pseudoalcaligenes, Pseudomonas pseudoflava, Pseudomonas putida,Pseudomonas putida var. naraensis, Pseudomonas putrefaciens, Pseudomonaspyrrocinia, Pseudomonas radiora, Pseudomonas reptilivora, Pseudomonasrhodesiae, Pseudomonas rhodos, Pseudomonas riboflavina, Pseudomonasrubescens, Pseudomonas rubrisubalbicans, Pseudomonas ruhlandii,Pseudomonas saccharophila, Pseudomonas savastanoi, Pseudomonassavastanoi pvar. glycinea, Pseudomonas savastanoi pvar. phaseolicola,Pseudomonas solanacearum, Pseudomonas sp., Pseudomonas spinosa,Pseudomonas stanieri, Pseudomonas stutzeri, Pseudomonas syringae,Pseudomonas syringae pvar. aptata, Pseudomonas syringae pvar.atrofaciens, Pseudomonas syringae pvar. coronafaciens, Pseudomonassyringae pvar. delphinii, Pseudomonas syringae pvar. glycinea,Pseudomonas syringae pvar. helianthi, Pseudomonas syringae pvar.lachrymans, Pseudomonas syringae pvar. lapsa, Pseudomonas syringae pvar.morsprunorum, Pseudomonas syringae pvar. phaseolicola, Pseudomonassyringae pvar. primulae, Pseudomonas syringae pvar. syringae,Pseudomonas syringae pvar. tabaci, Pseudomonas syringae pvar. tomato,Pseudomonas syringae subsp. glycinea, Pseudomonas syringae subsp.savastanoi, Pseudomonas syringae subsp. syringae, Pseudomonas syzygii,Pseudomonas tabaci, Pseudomonas taeniospiralis, Pseudomonastestosteroni, Pseudomonas thermocarboxydovorans, Pseudomonasthermotolerans, Pseudomonas thivervalensis, Pseudomonas tomato,Pseudomonas trivialis, Pseudomonas veronii, Pseudomonas vesicularis,Pseudomonas viridiflava, Pseudomonas viscogena, Pseudomonas woodsii,Rhizobacter dauci, Rhizobacter daucus or Xylophilus ampelinus;Rhizobiaceae such as the genera Agrobacterium, Carbophilus,Chelatobacter, Ensifer, Rhizobium, Sinorhizobium e.g. the speciesAgrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacteriumgelatinovorum, Agrobacterium lanymoorei, Agrobacterium meteori,Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium rubi,Agrobacterium stellulatum, Agrobacterium tumefaciens, Agrobacteriumvitis, Carbophilus carboxidus, Chelatobacter heintzii, Ensiferadhaerens, Ensifer arboris, Ensifer fredii, Ensifer kostiensis, Ensiferkummerowiae, Ensifer medicae, Ensifer meliloti, Ensifer saheli, Ensiferterangae, Ensifer xinjiangensis, Rhizobium ciceri Rhizobium etli,Rhizobium fredii, Rhizobium galegae, Rhizobium gallicum, Rhizobiumgiardinii, Rhizobium hainanense, Rhizobium huakuii, Rhizobiumhuautlense, Rhizobium indigoferae, Rhizobium japonicum, Rhizobiumleguminosarum, Rhizobium loessense, Rhizobium loti, Rhizobium lupini,Rhizobium mediterraneum, Rhizobium meliloti, Rhizobium mongolense,Rhizobium phaseoli, Rhizobium radiobacter, Rhizobium rhizogenes,Rhizobium rubi, Rhizobium sullae, Rhizobium tianshanense, Rhizobiumtrifolii, Rhizobium tropici, Rhizobium undicola, Rhizobium vitis,Sinorhizobium adhaerens, Sinorhizobium arboris, Sinorhizobium fredii,Sinorhizobium kostiense, Sinorhizobium kummerowiae, Sinorhizobiummedicae, Sinorhizobium meliloti, Sinorhizobium morelense, Sinorhizobiumsaheli or Sinorhizobium xinjiangense; Streptomycetaceae such as thegenera Kitasatosprora, Streptomyces, Streptoverticillium e.g. thespecies Streptomyces abikoensis, Streptomyces aburaviensis, Streptomycesachromogenes subsp. achromogenes, Streptomyces achromogenes subsp.rubradiris, Streptomyces acidiscabies, Streptomyces acrimycini,Streptomyces aculeolatus, Streptomyces afghaniensis, Streptomycesalanosinicus, Streptomyces albaduncus, Streptomyces albiaxialis,Streptomyces albidochromogenes, Streptomyces albidoflavus, Streptomycesalbireticuli, Streptomyces albofaciens, Streptomyces alboflavus,Streptomyces albogriseolus, Streptomyces albolongus, Streptomycesalboniger, Streptomyces albospinus, Streptomyces albosporeus subsp.albosporeus, Streptomyces albosporeus subsp. labilomyceticus,Streptomyces alboverticillatus, Streptomyces albovinaceus, Streptomycesalboviridis, Streptomyces albulus, Streptomyces albus subsp. albus,Streptomyces albus subsp. pathocidicus, Streptomyces almquistii,Streptomyces althioticus, Streptomyces amakusaensis, Streptomycesambofaciens, Streptomyces aminophilus, Streptomyces anandii,Streptomyces anthocyanicus, Streptomyces antibioticus, Streptomycesantimycoticus, Streptomyces anulatus, Streptomyces arabicus,Streptomyces ardus, Streptomyces arenae, Streptomyces argenteolus,Streptomyces armeniacus, Streptomyces asiaticus, Streptomycesasterosporus, Streptomyces atratus, Streptomyces atroaurantiacus,Streptomyces atroolivaceus, Streptomyces atrovirens, Streptomycesaurantiacus, Streptomyces aurantiogriseus, Streptomyces aureocirculatus,Streptomyces aureofaciens, Streptomyces aureorectus, Streptomycesaureoversilis, Streptomyces aureoverticillatus, Streptomyces aureus,Streptomyces avellaneus, Streptomyces avermectinius, Streptomycesavermitilis, Streptomyces avidinii, Streptomyces azaticus, Streptomycesazureus, Streptomyces baarnensis, Streptomyces bacillaris, Streptomycesbadius, Streptomyces baldaccii, Streptomyces bambergiensis, Streptomycesbejiangensis, Streptomyces bellus, Streptomyces bikiniensis,Streptomyces biverticillatus, Streptomyces blastmyceticus, Streptomycesbluensis, Streptomyces bobili, Streptomyces bottropensis, Streptomycesbrasiliensis, Streptomyces bungoensis, Streptomyces cacaoi subsp.asoensis, Streptomyces cacaoi subsp. cacaoi, Streptomyces caelestis,Streptomyces caeruleus, Streptomyces californicus, Streptomyces calvus,Streptomyces canaries, Streptomyces candidus, Streptomyces canescens,Streptomyces cangkringensis, Streptomyces caniferus, Streptomyces canus,Streptomyces capillispiralis, Streptomyces capoamus, Streptomycescarpaticus, Streptomyces carpinensis, Streptomyces catenulae,Streptomyces caviscabies, Streptomyces cavourensis subsp. cavourensis,Streptomyces cavourensis subsp. washingtonensis, Streptomycescellostaticus, Streptomyces celluloflavus, Streptomyces cellulolyticus,Streptomyces cellulosae, Streptomyces champavatii, Streptomyceschartreuses, Streptomyces chattanoogensis, Streptomyces chibaensis,Streptomyces chrestomyceticus, Streptomyces chromofuscus, Streptomyceschryseus, Streptomyces chrysomallus subsp. chrysomallus, Streptomyceschrysomallus subsp. fumigatus, Streptomyces cinereorectus, Streptomycescinereoruber subsp. cinereoruber, Streptomyces cinereoruber subsp.fructofermentans, Streptomyces cinereospinus, Streptomyces cinereus,Streptomyces cinerochromogenes, Streptomyces cinnabarinus, Streptomycescinnamonensis, Streptomyces cinnamoneus, Streptomyces cinnamoneus subsp.albosporus, Streptomyces cinnamoneus subsp. cinnamoneus, Streptomycescinnamoneus subsp. lanosus, Streptomyces cinnamoneus subsp. sparsus,Streptomyces cirratus, Streptomyces ciscaucasicus, Streptomycescitreofluorescens, Streptomyces clavifer, Streptomyces clavuligerus,Streptomyces cochleatus, Streptomyces coelescens, Streptomycescoelicoflavus, Streptomyces coelicolor, Streptomyces coeruleoflavus,Streptomyces coeruleofuscus, Streptomyces coeruleoprunus, Streptomycescoeruleorubidus, Streptomyces coerulescens, Streptomyces collinus,Streptomyces colombiensis, Streptomyces corchorusii, Streptomycescostaricanus, Streptomyces cremeus, Streptomyces crystallinus,Streptomyces curacoi, Streptomyces cuspidosporus, Streptomycescyaneofuscatus, Streptomyces cyaneus, Streptomyces cyanoalbus,Streptomyces cystargineus, Streptomyces daghestanicus, Streptomycesdiastaticus subsp. ardesiacus, Streptomyces diastaticus subsp.diastaticus, Streptomyces diastatochromogenes, Streptomyces distallicus,Streptomyces djakartensis, Streptomyces durhamensis, Streptomycesechinatus, Streptomyces echinoruber, Streptomyces ederensis,Streptomyces ehimensis, Streptomyces endus, Streptomyces enissocaesilis,Streptomyces erumpens, Streptomyces erythraeus, Streptomyceserythrogriseus, Streptomyces eurocidicus, Streptomyces europaeiscabiei,Streptomyces eurythermus, Streptomyces exfoliates, Streptomyces felleus,Streptomyces fervens, Streptomyces fervens subsp. fervens, Streptomycesfervens subsp. melrosporus, Streptomyces flamentosus, Streptomycesfilipinensis, Streptomyces fimbriatus, Streptomyces fimicarius,Streptomyces finlayi, Streptomyces flaveolus, Streptomyces flaveus,Streptomyces flavidofuscus, Streptomyces flavidovirens, Streptomycesflaviscleroticus, Streptomyces flavofungini, Streptomyces flavofuscus,Streptomyces flavogriseus, Streptomyces flavopersicus, Streptomycesflavotricini, Streptomyces flavovariabilis, Streptomyces flavovirens,Streptomyces flavoviridis, Streptomyces flocculus, Streptomycesfloridae, Streptomyces fluorescens, Streptomyces fradiae, Streptomycesfragilis, Streptomyces fulvissimus, Streptomyces fulvorobeus,Streptomyces fumanus, Streptomyces fumigatiscleroticus, Streptomycesgalbus, Streptomyces galilaeus, Streptomyces gancidicus, Streptomycesgardneri, Streptomyces gelaticus, Streptomyces geysiriensis,Streptomyces ghanaensis, Streptomyces gibsonii, Streptomycesglaucescens, Streptomyces glaucosporus, Streptomyces glaucus,Streptomyces globisporus subsp. caucasicus, Streptomyces globisporussubsp. flavofuscus, Streptomyces globisporus subsp. globisporus,Streptomyces globosus, Streptomyces glomeratus, Streptomycesglomeroaurantiacus, Streptomyces gobitricini, Streptomyces goshikiensis,Streptomyces gougerotii, Streptomyces graminearus, Streptomycesgraminofaciens, Streptomyces griseinus, Streptomyces griseoaurantiacus,Streptomyces griseobrunneus, Streptomyces griseocameus, Streptomycesgriseochromogenes, Streptomyces griseoflavus, Streptomyces griseofuscus,Streptomyces griseoincamratus, Streptomyces griseoloalbus, Streptomycesgriseolosporeus, Streptomyces griseolus, Streptomyces griseoluteus,Streptomyces griseomycini, Streptomyces griseoplanus, Streptomycesgriseorubens, Streptomyces griseoruber, Streptomyces griseorubiginosus,Streptomyces griseosporeus, Streptomyces griseostramineus, Streptomycesgriseoverticillatus, Streptomyces griseoviridis, Streptomyces griseussubsp. alpha, Streptomyces griseus subsp. cretosus, Streptomyces griseussubsp. griseus, Streptomyces griseus subsp. solvifaciens, Streptomyceshachijoensis, Streptomyces halstedii, Streptomyces hawaiiensis,Streptomyces heliomycini, Streptomyces helvaticus, Streptomycesherbaricolor, Streptomyces hiroshimensis, Streptomyces hirsutus,Streptomyces humidus, Streptomyces humiferus, Streptomyces hydrogenans,Streptomyces hygroscopicus subsp. angustmyceticus, Streptomyceshygroscopicus subsp. decoyicus, Streptomyces hygroscopicus subsp.glebosus, Streptomyces hygroscopicus subsp. hygroscopicus, Streptomyceshygroscopicus subsp. ossamyceticus, Streptomyces iakyrus, Streptomycesindiaensis, Streptomyces indigoferus, Streptomyces indonesiensis,Streptomyces intermedius, Streptomyces inusitatus, Streptomycesipomoeae, Streptomyces janthinus, Streptomyces javensis, Streptomyceskanamyceticus, Streptomyces kashmirensis, Streptomyces kasugaensis,Streptomyces katrae, Streptomyces kentuckensis, Streptomyces kifunensis,Streptomyces kishiwadensis, Streptomyces kunmingensis, Streptomyceskurssanovii, Streptomyces labedae, Streptomyces laceyi, Streptomycesladakanum, Streptomyces lanatus, Streptomyces lateritius, Streptomyceslaurentii, Streptomyces lavendofoliae, Streptomyces lavendulae subsp.grasserius, Streptomyces lavendulae subsp. lavendulae, Streptomyceslavenduligriseus, Streptomyces lavendulocolor, Streptomyces levis,Streptomyces libani subsp. libani, Streptomyces libani subsp. rufus,Streptomyces lienomycini, Streptomyces lilacinus, Streptomyces limosus,Streptomyces lincolnensis, Streptomyces lipmanii, Streptomyceslitmocidini, Streptomyces lomondensis, Streptomyces longisporoflavus,Streptomyces longispororuber, Streptomyces longisporus, Streptomyceslongwoodensis, Streptomyces lucensis, Streptomyces luridiscabiei,Streptomyces luridus, Streptomyces lusitanus, Streptomycesluteireticuli, Streptomyces luteogriseus, Streptomyces luteosporeus,Streptomyces luteoverticillatus, Streptomyces lydicus, Streptomycesmacrosporus, Streptomyces malachitofuscus, Streptomyces malachitospinus,Streptomyces malaysiensis, Streptomyces mashuensis, Streptomycesmassasporeus, Streptomyces matensis, Streptomyces mauvecolor,Streptomyces mediocidicus, Streptomyces mediolani, Streptomycesmegasporus, Streptomyces melanogenes, Streptomyces melanosporofaciens,Streptomyces mexicanus, Streptomyces michiganensis, Streptomycesmicroflavus, Streptomyces minutiscleroticus, Streptomyces mirabilis,Streptomyces misakiensis, Streptomyces misionensis, Streptomycesmobaraensis, Streptomyces monomycini, Streptomyces morookaensis,Streptomyces murinus, Streptomyces mutabilis, Streptomyces mutomycini,Streptomyces naganishii, Streptomyces narbonensis, Streptomycesnashvillensis, Streptomyces netropsis, Streptomyces neyagawaensis,Streptomyces niger, Streptomyces nigrescens, Streptomyces nigrifaciens,Streptomyces nitrosporeus, Streptomyces niveiciscabiei, Streptomycesniveoruber, Streptomyces niveus, Streptomyces noboritoensis,Streptomyces nodosus, Streptomyces nogalater, Streptomyces nojiriensis,Streptomyces noursei, Streptomyces novaecaesareae, Streptomycesochraceiscleroticus, Streptomyces odorifer, Streptomycesolivaceiscleroticus, Streptomyces olivaceoviridis, Streptomycesolivaceus, Streptomyces olivochromogenes, Streptomyces olivomycini,Streptomyces olivoreticuli, Streptomyces olivoreticuli subsp.cellulophilus, Streptomyces olivoreticuli subsp. olivoreticuli,Streptomyces olivoverticillatus, Streptomyces olivoviridis, Streptomycesomiyaensis, Streptomyces orinoci, Streptomyces pactum, Streptomycesparacochleatus, Streptomyces paradoxus, Streptomyces parvisporogenes,Streptomyces parvulus, Streptomyces parvus, Streptomyces peucetius,Streptomyces phaeochromogenes, Streptomyces phaeofaciens, Streptomycesphaeopurpureus, Streptomyces phaeoviridis, Streptomyces phosalacineus,Streptomyces pilosus, Streptomyces platensis, Streptomyces plicatus,Streptomyces pluricolorescens, Streptomyces polychromogenes,Streptomyces poonensis, Streptomyces praecox, Streptomycesprasinopilosus, Streptomyces prasinosporus, Streptomyces prasinus,Streptomyces prunicolor, Streptomyces psammoticus, Streptomycespseudoechinosporeus, Streptomyces pseudogriseolus, Streptomycespseudovenezuelae, Streptomyces pulveraceus, Streptomyces puniceus,Streptomyces puniciscabiei, Streptomyces purpeofuscus, Streptomycespurpurascens, Streptomyces purpureus, Streptomycespurpurogeneiscleroticus, Streptomyces racemochromogenes, Streptomycesrameus, Streptomyces ramuilosus, Streptomyces rangoonensis, Streptomycesrecifensis, Streptomyces rectiverticillatus, Streptomycesrectiviolaceus, Streptomyces regensis, Streptomyces resistomycificus,Streptomyces reticuliscabiei, Streptomyces rhizosphaericus, Streptomycesrimosus subsp. paromomycinus, Streptomyces rimosus subsp. rimosus,Streptomyces rishiriensis, Streptomyces rochei, Streptomycesroseiscleroticus, Streptomyces roseodiastaticus, Streptomycesroseoflavus, Streptomyces roseofulvus, Streptomyces roseolilacinus,Streptomyces roseolus, Streptomyces roseosporus, Streptomycesroseoverticillatus, Streptomyces roseoviolaceus, Streptomycesroseoviridis, Streptomyces rubber, Streptomyces rubiginosohelvolus,Streptomyces rubiginosus, Streptomyces rubrogriseus, Streptomycesrutgersensis subsp. castelarensis, Streptomyces rutgersensis subsp.rutgersensis, Streptomyces salmonis, Streptomyces sampsonii,Streptomyces sanglieri, Streptomyces sannanensis, Streptomycessapporonensis, Streptomyces scabiei, Streptomyces sclerotialus,Streptomyces scopiformis, Streptomyces seoulensis, Streptomycesseptatus, Streptomyces setae, Streptomyces setonii, Streptomycesshowdoensis, Streptomyces sindenensis, Streptomyces sioyaensis,Streptomyces somaliensis, Streptomyces sparsogenes, Streptomycesspectabilis, Streptomyces speibonae, Streptomyces speleomycini,Streptomyces spheroids, Streptomyces spinoverrucosus, Streptomycesspiralis, Streptomyces spiroverticillatus, Streptomyces spitsbergensis,Streptomyces sporocinereus, Streptomyces sporoclivatus, Streptomycesspororaveus, Streptomyces sporoverrucosus, Streptomyces stelliscabiei,Streptomyces stramineus, Streptomyces subrutilus, Streptomycessulfonofaciens, Streptomyces sulphurous, Streptomyces syringium,Streptomyces tanashiensis, Streptomyces tauricus, Streptomyces tendae,Streptomyces termitum, Streptomyces thermoalcalitolerans, Streptomycesthermoautotrophicus, Streptomyces thermocarboxydovorans, Streptomycesthermocarboxydus, Streptomyces thermocoprophilus, Streptomycesthermodiastaticus, Streptomyces thermogriseus, Streptomycesthermolineatus, Streptomyces thermonitrifcans, Streptomycesthermospinosisporus, Streptomyces thermoviolaceus subsp. apingens,Streptomyces thermoviolaceus subsp. thermoviolaceus, Streptomycesthermovulgaris, Streptomyces thioluteus, Streptomyces torulosus,Streptomyces toxytricini, Streptomyces tricolor, Streptomycestubercidicus, Streptomyces tuirus, Streptomyces turgidiscabies,Streptomyces umbrinus, Streptomyces variabilis, Streptomyces variegates,Streptomyces varsoviensis, Streptomyces vastus, Streptomyces venezuelae,Streptomyces vinaceus, Streptomyces vinaceusdrappus, Streptomycesviolaceochromogenes, Streptomyces violaceolatus, Streptomycesviolaceorectus, Streptomyces violaceoruber, Streptomycesviolaceorubidus, Streptomyces violaceus, Streptomyces violaceusniger,Streptomyces violarus, Streptomyces violascens, Streptomyces violatus,Streptomyces violens, Streptomyces virens, Streptomyces virginiae,Streptomyces viridiflavus, Streptomyces viridiviolaceus, Streptomycesviridobrunneus, Streptomyces viridochromogenes, Streptomycesviridodiastaticus, Streptomyces viridosporus, Streptomycesvitaminophileus, Streptomyces vitaminophilus, Streptomyces wedmorensis,Streptomyces werraensis, Streptomyces willmorei, Streptomycesxanthochromogenes, Streptomyces xanthocidicus, Streptomycesxantholiticus, Streptomyces xanthophaeus, Streptomyces yatensis,Streptomyces yerevanensis, Streptomyces yogyakartensis, Streptomycesyokosukanensis, Streptomyces yunnanensis, Streptomyces zaomyceticus,Streptoverticillium abikoense, Streptoverticillium albireticuli,Streptoverticillium alboverticillatum, Streptoverticillium album,Streptoverticillium ardum, Streptoverticillium aureoversale,Streptoverticillium aureoversile, Streptoverticillium baldaccii,Streptoverticillium biverticillatum, Streptoverticillium blastmyceticum,Streptoverticillium cinnamoneum subsp. albosporum, Streptomycescinnamoneus subsp. albosporus, Streptoverticillium cinnamoneum subsp.cinnamoneum, Streptoverticillium cinnamoneum subsp. lanosum,Streptoverticillium cinnamoneum subsp. sparsum, Streptoverticilliumdistallicum, Streptoverticillium ehimense, Streptoverticilliumeurocidicum, Streptoverticillium fervens subsp. fervens,Streptoverticillium fervens subsp. melrosporus, Streptoverticilliumflavopersicum, Streptoverticillium griseocarneum, Streptoverticilliumgriseoverticillatum, Streptoverticillium hachijoense,Streptoverticillium hiroshimense, Streptoverticillium kashmirense,Streptoverticillium kentuckense, Streptoverticillium kishiwadense,Streptoverticillium ladakanum, Streptoverticillium lavenduligriseum,Streptoverticillium lilacinum, Streptoverticillium luteoverticillatum,Streptoverticillium mashuense, Streptoverticillium mobaraense,Streptoverticillium morookaense, Streptoverticillium netropsis,Streptoverticillium olivomycini, Streptomyces olivomycini,Streptoverticillium olivoreticuli subsp. cellulophilum,Streptoverticillium olivoreticuli subsp. olivoreticuli,Streptoverticillium olivoreticulum, Streptoverticillium olivoreticulumsubsp. cellulophilum, Streptoverticillium olivoverticillatum,Streptoverticillium orinoci, Streptoverticillium parvisporogenes,Streptoverticillium parvisporogenum, Streptoverticilliumrectiverticillatum, Streptoverticillium reticulum subsp. protomycicum,Streptoverticillium roseoverticillatum, Streptoverticillium salmonis,Streptoverticillium sapporonense, Streptovefticillium septatum,Streptoverticillium syringium, Streptoverticillium thioluteum,Streptoverticillium verticillium subsp. quantum, Streptoverticilliumverticillium subsp. tsukushiense or Streptoverticillium viridoflavum.

Particular preferred strains are strains selected from the groupconsisting of Bacillaceae, Brevibacteriaceae, Corynebacteriaceae,Nocardiaceae, Mycobacteriaceae, Streptomycetaceae, Enterobacteriaceaesuch as Bacillus circulans, Bacillus subtilis, Bacillus sp.,Brevibacterium albidum, Brevibacterium album, Brevibacterium cerinum,Brevibacterium flavum, Brevibacterium glutamigenes, Brevibacteriumiodinum, Brevibacterium ketoglutamicum, Brevibacterium lactofermentum,Brevibacterium linens, Brevibacterium roseum, Brevibacteriumsaccharolyticum, Brevibacterium sp., Corynebacterium acetoacidophilum,Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes,Corynebacterium glutamicum (=Micrococcus glutamicum), Corynebacteriummelassecola, Corynebacterium sp., Nocardia rhodochrous (Rhodococcusrhodochrous), Mycobacterium rhodochrous, Streptomyces lividans andEscherichia coli especially Escherichia coli K12.

In addition particular preferred strains are strains selected from thegroup consisting of Cryptococcaceae, Saccharomycetaceae,Schizosaccharomycetacease such as the genera Candida, Hansenula, Pichia,Saccharomyces and Schizosaccharomyces preferred are strains selectedfrom the group consisting of the species Rhodotorula rubra, Rhodotorulaglutinis, Rhodotorula graminis, Yarrowia lipolytica, Sporobolomycessalmonicolor, Sporobolomyces shibatanus, Saccharomyces cerevisiae,Candida boidinii, Candida bombicola, Candida cylindracea, Candidaparapsilosis, Candida rugosa, Candida tropicalis, Pichia methanolica andPichia pastoris especially Saccharomyces cerevisiae.

Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium e.g.the species Pistacia vera [pistachios, Pistazie], Mangifer indica[Mango] or Anacardium occidentale [Cashew]; Asteraceae such as thegenera Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus,Lactuca, Locusta, Tagetes, Valeriana e.g. the species Calendulaofficinalis [Marigold], Carthamus tinctorius [safflower], Centaureacyanus [cornflower], Cichorium intybus [blue daisy], Cynara scolymus[Artichoke], Helianthus annus [sunflower], Lactuca sativa, Lactucacrispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa, Lactucascariola L. var. integrata, Lactuca scariola L. var. integrifolia,Lactuca sativa subsp. romana, Locusta communis, Valeriana locusta[lettuce], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia[Marigold]; Apiaceae such as the genera Daucus e.g. the species Daucuscarota [carrot]; Betulaceae such as the genera Corylus e.g. the speciesCorylus avellana or Corylus colurna [hazelnut]; Boraginaceae such as thegenera Borago e.g. the species Borago officinalis [borage]; Brassicaceaesuch as the genera Brassica, Melanosinapis, Sinapis, Arabadopsis e.g.the species Brassica napus, Brassica rapa ssp. [canola, oilseed rape,turnip rape], Sinapis arvensis, Brassica juncea, Brassica juncea var.juncea, Brassica juncea var. crispifolia, Brassica juncea var. foliosa,Brassica nigra, Brassica sinapioides, Melanosinapis communis [mustard],Brassica oleracea [fodder beet] or Arabidopsis thaliana; Bromeliaceaesuch as the genera Anana, Bromelia e.g. the species Anana comosus,Ananas ananas or Bromelia comosa [pineapple]; Caricaceae such as thegenera Carica e.g. the species Carica papaya [papaya]; Cannabaceae suchas the genera Cannabis e.g. the species Cannabis sative [hemp],Convolvulaceae such as the genera Ipomea, Convolvulus e.g. the speciesIpomoea batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulustiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba orConvolvulus panduratus [sweet potato, Man of the Earth, wild potato],Chenopodiaceae such as the genera Beta, i.e. the species Beta vulgaris,Beta vulgaris var. altissima, Beta vulgaris var. Vulgaris, Betamaritima, Beta vulgaris var. perennis, Beta vulgaris var. conditiva orBeta vulgaris var. esculenta [sugar beet]; Cucurbitaceae such as thegenera Cucurbita e.g. the species Cucurbita maxima, Cucurbita mixta,Cucurbita pepo or Cucurbita moschata [pumpkin, squash]; Elaeagnaceaesuch as the genera Elaeagnus e.g. the species Olea europaea [olive];Ericaceae such as the genera Kalmia e.g. the species Kalmia latifolia,Kalmia angustifolia, Kalmia microphylla, Kalmia polifolia, Kalmiaoccidentalis, Cistus chamaerhodendros or Kalmia lucida [American laurel,broad-leafed laurel, calico bush, spoon wood, sheep laurel, alpinelaurel, bog laurel, western bog-laurel, swamp-laurel]; Euphorbiaceaesuch as the genera Manihot, Janipha, Jatropha, Ricinus e.g. the speciesManihot utilissima, Janipha manihot, Jatropha manihot, Manihot aipil,Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta[manihot, arrowroot, tapioca, cassava] or Ricinus communis [castor bean,Castor Oil Bush, Castor Oil Plant, Palma Christi, Wonder Tree]; Fabaceaesuch as the genera Pisum, Albizia, Cathormion, Feuillea, Inga,Pithecolobium, Acacia, Mimosa, Medicajo, Glycine, Dolichos, Phaseolus,Soja e.g. the species Pisum sativum, Pisum arvense, Pisum humile [pea],Albizia berteriana, Albizia julibrissin, Albizia lebbeck, Acaciaberteriana, Acacia littoralis, Albizia berteriana, Albizzia berteriana,Cathormion berteriana, Feuillea berteriana, Inga fragrans,Pithecellobium berterianum, Pithecellobium fragrans, Pithecolobiumberterianum, Pseudalbizzia berteriana, Acacia julibrissin, Acacia nemu,Albizia nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosaspeciosa, Sericanrda julibrissin, Acacia lebbeck, Acacia macrophylla,Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa[bastard logwood, silk tree, East Indian Walnut], Medicago sativa,Medicago falcata, Medicago varia [alfalfa] Glycine max Dolichos soja,Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida or Sojamax [soybean]; Geraniaceae such as the genera Pelargonium, Cocos, Oleume.g. the species Cocos nucifera, Pelargonium grossularioides or Oleumcocois [coconut]; Gramineae such as the genera Saccharum e.g. thespecies Saccharum officinarum; Juglandaceae such as the genera Juglans,Wallia e.g. the species Juglans regia, Juglans ailanthifolia, Juglanssieboldiana, Juglans cinerea, Wallia cinerea, Juglans bixbyi, Juglanscalifornica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis,Juglans major, Juglans microcarpa, Juglans nigra or Wallia nigra[walnut, black walnut, common walnut, persian walnut, white walnut,butternut, black walnut]; Lauraceae such as the genera Persea, Lauruse.g. the species laurel Laurus nobilis [bay, laurel, bay laurel, sweetbay], Persea americana, Persea americana, Persea gratissima or Perseapersea [avocado]; Leguminosae such as the genera Arachis e.g. thespecies Arachis hypogaea [peanut]; Linaceae such as the genera Linum,Adenolinum e.g. the species Linum usitatissimum, Linum humile, Linumaustriacum, Linum bienne, Linum angustifolium, Linum catharticum, Linumflavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii,Linum narbonense, Linum perenne, Linum perenne var. lewisii, Linumpratense or Linum trigynum [flax, linseed]; Lythrarieae such as thegenera Punica e.g. the species Punica granatum [pomegranate]; Malvaceaesuch as the genera Gossypium e.g. the species Gossypium hirsutum,Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum orGossypium thurberi [cotton]; Musaceae such as the genera Musa e.g. thespecies Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [banana];Onagraceae such as the genera Camissonia, Oenothera e.g. the speciesOenothera biennis or Camissonia brevipes [primrose, evening primrose];Palmae such as the genera Elacis e.g. the species Elaeis guineensis [oilplam]; Papaveraceae such as the genera Papaver e.g. the species Papaverorientale, Papaver rhoeas, Papaver dubium [poppy, oriental poppy, cornpoppy, field poppy, shirley poppies, field poppy, long-headed poppy,long-pod poppy]; Pedaliaceae such as the genera Sesamum e.g. the speciesSesamum indicum [sesame]; Piperaceae such as the genera Piper, Artanthe,Peperomia, Steffensia e.g. the species Piper aduncum, Piper amalago,Piper angustifolium, Piper auritum, Piper betel, Piper cubeba, Piperlongum, Piper nigrum, Piper retrofractum, Artanthe adunca, Artantheelongata, Peperomia elongata, Piper elongatum, Steffensia elongata.[Cayenne pepper, wild pepper]; Poaceae such as the genera Hordeum,Secale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea,Triticum e.g. the species Hordeum vulgare, Hordeum jubatum, Hordeummurinum, Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeumhexastichon, Hordeum hexastichum, Hordeum irregulare, Hordeum sativum,Hordeum secalinum [barley, pearl barley, foxtail barley, wall barley,meadow barley], Secale cereale [rye], Avena sativa, Avena fatua, Avenabyzantina, Avena fatua var. sativa, Avena hybrida [oat], Sorghumbicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgare,Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghumaethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum,Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghumsubglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcushalepensis, Sorghum miliaceum millet, Panicum militaceum [Sorghum,millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn, maize]Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum,Triticum macha, Triticum sativum or Triticum vulgare [wheat, breadwheat, common wheat], Proteaceae such as the genera Macadamia e.g. thespecies Macadamia intergrifolia [macadamia); Rubiaceae such as thegenera Coffea e.g. the species Coffea spp., Coffea arabica, Coffeacanephora or Coffea liberica [coffee]; Scrophulariaceae such as thegenera Verbascum e.g. the species Verbascum blattaria, Verbascumchaixii, Verbascum densiflorum, Verbascum lagurus, Verbascumlongifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum,Verbascum phlomoides, Verbascum phoenicum, Verbascum pulverulentum orVerbascum thapsus [mullein, white moth mullein, nettle-leaved mullein,dense-flowered mullein, silver mullein, long-leaved mullein, whitemullein, dark mullein, greek mullein, orange mullein, purple mullein,hoary mullein, great mullein]; Solanaceae such as the genera Capsicum,Nicotiana, Solanum, Lycopersicon e.g. the species Capsicum annuum,Capsicum annuum var. glabriusculum, Capsicum frutescens [pepper],Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata, Nicotianaattenuata, Nicotiana glauca, Nicotiana langsdorffi, Nicotianaobtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotianarustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [potato],Solanum melongena [egg-plant] (Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanumlycopersicum [tomato]; Sterculiaceae such as the genera Theobroma e.g.the species Theobroma cacao [cacao]; Theaceae such as the generaCamellia e.g. the species Camellia sinensis) [tea]. All abovementionedorganisms can in princible also function as host organisms.

Particular preferred plants are plants selected from the groupconsisting of Asteraceae such as the genera Helianthus, Tagetes e.g. thespecies Helianthus annus [sunflower], Tagetes lucida, Tagetes erecta orTagetes tenuifolia [Marigold]; Brassicaceae such as the genera Brassica,Arabadopsis e.g. the species Brassica napus, Brassica rapa ssp.,Brassica juncea [canola, oilseed rape, turnip rape] or Arabidopsisthaliana; Fabaceae such as the genera Glycine e.g. the species Glycinemax, Soja hispida or Soja max [soybean]; Linaceae such as the generaLinum e.g. the species Linum usitatissimum, [flax, linseed]; Poaceaesuch as the genera Hordeum, Secale, Avena, Sorghum, Oryza, Zea, Triticume.g. the species Hordeum vulgare [barley]; Secale cereale [rye], Avenasativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avenahybrida [oat], Sorghum bicolor [Sorghum, millet], Oryza sativa, Oryzalatifolia [rice], Zea mays [corn, maize] Triticum aestivum, Triticumdurum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticumsativum or Triticum vulgare [wheat, bread wheat, common wheat];Solanaceae such as the genera Solanum, Lycopersicon e.g. the speciesSolanum tuberosum [potato], Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanumlycopersicum [tomato].

All abovementioned organisms can in princible also function as hostorganisms.

With regard to the nucleic acid sequence as depicted below a nucleicacid construct which contains a nucleic acid sequence mentioned hereinor an organism (=transgenic organism) which is transformed with saidnucleic acid sequence or said nucleic acid construct, “transgene” meansall those constructs which have been brought about by geneticmanipulation methods, preferably in which either

-   a) the nucleic acid sequence as depicted in SEQ ID NO: 1, 3, 5, 7,    9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,    43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,    85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,    115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,    141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,    167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,    193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217,    219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,    245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269,    271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295,    297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,    323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,    349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373,    375, 377, 379, 381, 383, 385, 387, 389, 391 or 393 or a derivative    thereof, or-   b) a genetic regulatory element, for example a promoter, which is    functionally linked to the nucleic acid sequence as depicted in SEQ    ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,    33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,    75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,    107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,    133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,    159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,    185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,    211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,    237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,    263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287,    289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,    315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339,    341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365,    367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or    393 or a derivative thereof, or-   c) (a) and (b)    is/are not present in its/their natural genetic environment or    has/have been modified by means of genetic manipulation methods, it    being possible for the modification to be, by way of example, a    substitution, addition, deletion, inversion or insertion of one or    more nucleotide. “Natural genetic environment” means the natural    chromosomal locus in the organism of origin or the presence in a    genomic library. In the case of a genomic library, the natural,    genetic environment of the nucleic acid sequence is preferably at    least partially still preserved. The environment flanks the nucleic    acid sequence at least on one side and has a sequence length of at    least 50 bp, preferably at least 500 bp, particularly preferably at    least 1000 bp, very particularly preferably at least 5000 bp.

The use of the nucleic acid sequence according to the invention or ofthe nucleic acid construct according to the invention for the generationof transgenic plants is therefore also subject matter of the invention.

The fine chemical, which is synthesized in the organism, in particularthe microorganism, the cell, the tissue or the plant, of the inventioncan be isolated if desired. Depending on the use of the fine chemical,different purities resulting from the purification may be advantageousas will be described herein below.

In an advantageous embodiment of the invention, the organism takes theform of a plant whose fine chemical content is modified advantageouslyowing to the nucleic acid molecule of the present invention expressed.This is important for plant breeders since, for example, the nutritionalvalue of organisms such as a plant is very often limited by its aminoacid, protein, co-factor and/or vitamin content to mention only a coupleof them. For example in feed for monogastric animals a few essentialamino acids such as lysine, threonine or methionine are very oftenlimiting. After the biological activity of the nucleic acid and/orprotein of the invention has been increased or generated, or after theexpression of nucleic acid molecule or polypeptide according to theinvention has been generated or increased, the transgenic plantgenerated thus is grown on or in a nutrient medium or else in the soiland subsequently harvested.

The plants or parts thereof, e.g. the leaves, roots, flowers, and/orstems and/or other harvestable material as described below, can then beused directly as foodstuffs or animal feeds or else be furtherprocessed. Again, the amino acids can be purified further in thecustomary manner via extraction and precipitation or via ion exchangersand other methods known to the person skilled in the art and describedherein below. Products which are suitable for various applications andwhich result from these different processing procedures are for exampleamino acids or amino acid compositions which can still comprise furtherplant components in different amounts, advantageously in the range offrom 0 to 99% by weight or more, preferably from below 90%, 80%, 70%,60% or 50% by weight, especially preferably below 40%, 30%, 20% or 10%by weight. The plants can also advantageously be used directly withoutfurther processing, e.g. as feed or for extraction.

The chemically pure fine chemical or chemically pure compositionscomprising the fine chemical may also be produced by the processdescribed above. To this end, the fine chemical or the compositions areisolated in the known manner from an organism according to theinvention, such as the microorganisms, non-human animal or the plants,and/or their culture medium in which or on which the organisms had beengrown. These chemically pure fine chemical or said compositions areadvantageous for applications in the field of the food industry, thecosmetics industry or the pharmaceutical industry.

Thus, the content of plant components and preferably also furtherimpurities is as low as possible, and the abovementioned fine chemicalare obtained in as pure form as possible. In these applications, thecontent of plant components advantageously amounts to less than 10% byweight, preferably 1% by weight, more preferably 0.1% by weight, veryespecially preferably 0.01% by weight or less.

Accordingly, the fine chemical produced by the present invention is atleast 0.1% by weight pure, preferably more than 1% by weight pure, morepreferred 10% by weight pure, even more preferred are more than 50%,60%, 70% or 80% by weight pure, even more preferred are more than 90%,91%, 92%, 93%, 94% or 95% weight pure, most preferred are 96%, 97%, 98%or 99% by weight or more pure.

In this context, the amount of the fine chemical in a cell of theinvention may be increased according to the process of the invention byat least a factor of 1.1, preferably at least a factor of 1.5; 2; or 5,especially preferably by at least a factor of 10 or 30, very especiallypreferably by at least a factor of 50, in comparison with the wild type,control or reference. Preferrably, said increase is found in a tissue ofan organism, more preferred in the organism itself or in a harvestablepart thereof.

In principle, the fine chemicals produced can be increased in two waysby the process according to the invention. The pool of free finechemicals, in particular of the free fine chemical, and/or the contentof bound for example protein-bound fine chemicals, in particular of theprotein-bound fine chemical may advantageously be increased.

It may be advantageous to increase the pool of free fine chemical in thetransgenic organisms by the process according to the invention in orderto isolate high amounts of the pure fine chemical.

In another preferred embodiment of the invention a combination of theincreased expression of the nucleic acid sequence or the protein of theinvention together with the transformation of a protein or polypeptid,which functions as a sink for the desired fine chemical such as an aminoacid for example methionine, lysine or threonine in the organism isuseful to increase the production of the fine chemical (see U.S. Pat.No. 5,589,616, WO 96/38574, WO 97/07665, WO 97/28247, U.S. Pat. No.4,886,878, U.S. Pat. No. 5,082,993 and U.S. Pat. No. 5,670,635). Galiliet al. [Transgenic Res. 2000] showed, that enhancing the synthesis ofthreonine by a feed back insensitive aspertate kinase did not lead onlyto in increase in free threonine but also in protein bound threonine.

In may also be advantageous to increase the content of the bound finechemical.

In a preferred embodiment, the fine chemical is produced in accordancewith the invention and, if desired, is isolated. The production offurther fine chemicals for example amino acids such as lysine and ofamino acid mixtures by the process according to the invention isadvantageous.

In the case of the fermentation of microorganisms, the abovementionedfine chemical e.g. amino acid or mixtures of amino acids may accumulatein the medium and/or the cells. If microorganisms are used in theprocess according to the invention, the fermentation broth can beprocessed after the cultivation. Depending on the requirement, all orsome of the biomass can be removed from the fermentation broth byseparation methods such as, for example, centrifugation, filtration,decanting or a combination of these methods, or else the biomass can beleft in the fermentation broth. The fermentation broth can subsequentlybe reduced, or concentrated, with the aid of known methods such as, forexample, rotary evaporator, thin-layer evaporator, falling filmevaporator, by reverse osmosis or by nanofiltration. This concentratedfermentation broth can subsequently be processed by lyophilization,spray drying, spray granulation or by other methods.

To purify a fine chemical such as an amino acid, a product-containingfermentation broth from which the biomass has been separated may besubjected to chromatography with a suitable resin such as ion exchangeresin for example anion or cation exchange resin, hydrophobic resin orhydrophilic resin for example epoxy resin, polyurethane resin orpolyacrylamid resin, or resin for separation according to the molecularweight of the compounds for example polyvinyl chloride homopolymer resinor resins composed for example of polymers of acrylic acid, crosslinkedwith polyalkenyl ethers or divinyl glycol such as Carbopol®, Pemulen®and Noveon®. If necessary these chromatography steps may be repeatedusing the same or other chromatography resins. The skilled worker isfamiliar with the choice of suitable chromatography resins and theirmost effective use. The purified product may be concentrated byfiltration or ultrafiltration and stored at a temperature, which ensuresthe maximum stability of the product.

The identity and purity of the compound(s) isolated can be determined byprior-art techniques. They encompass high-performance liquidchromatography (HPLC), spectroscopic methods, mass spectrometry (MS),staining methods, thin-layer chromatography, NIRS, enzyme assays ormicrobiological assays. These analytical methods are compiled in: Pateket al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al.(1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) BioprocessEngineer. 19:67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996)Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566,575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlasof Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.et al. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, vol. 17.

Fine chemicals like amino acids can for example be detectedadvantageously via HPLC separation in ethanolic extract as described byGeigenberger et al. (Plant Cell & Environ, 19, 1996: 43-55). Amino acidscan be extracted with hot water. After filtration the extracts arediluted with water containing 20 mg/mL sodium acide. The separation anddetection of the amino acids is performed using an anion exchange columnand an electrochemical detector. Technical details can be taken from Y.Ding et al., 2002, Direct determination of free amino acids and sugarsin green tea by anion-exchange chromatography with integrated pulsedamperometric detection, J Chromatogr A, (2002) 982; 237-244, or e.g.from Karchi et al., 1993, Plant J. 3: 721-727; Matthews M J, 1997(Lysine, threonine and methionine biosynthesis. In B K Singh, ed, PlantAmino Acids: Biochemistry and Biotechnology. Dekker, New York, pp205-225; H Hesse and R Hoefgen. (2003) Molecular aspects of methioninebiosynthesis. TIPS 8(259-262.

In a preferred embodiment, the present invention relates to a processfor the production of the fine chemical comprising or generating in anorganism or a part thereof the expression of at least one nucleic acidmolecule comprising a nucleic acid molecule selected from the groupconsisting of:

-   a) nucleic acid molecule encoding, preferably at least the mature    form, of the polypeptide as depicted in SEQ ID NO: 2, 4, 6, 8, 10,    12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,    46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,    88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,    118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,    144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,    170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,    196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,    222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246,    248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,    274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,    300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,    326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,    352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,    378, 380, 382, 384, 386, 388, 390, 392 or 394 or a fragment thereof,    which confers an increase in the amount of the fine chemical in an    organism or a part thereof;-   b) nucleic acid molecule comprising, preferably at least the mature    form, of the nucleic acid molecule as depicted in SEQ ID NO: 1, 3,    5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,    41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,    83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,    113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,    139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,    165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,    191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,    217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,    243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,    269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,    295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319,    321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345,    347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371,    373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393;-   c) nucleic acid molecule whose sequence can be deduced from a    polypeptide sequence encoded by a nucleic acid molecule of (a)    or (b) as result of the degeneracy of the genetic code and    conferring an increase in the amount of the fine chemical in an    organism or a part thereof;-   d) nucleic acid molecule encoding a polypeptide which has at least    50% identity with the amino acid sequence of the polypeptide encoded    by the nucleic acid molecule of (a) to (c) and conferring an    increase in the amount of the fine chemical in an organism or a part    thereof;-   e) nucleic acid molecule which hybidizes with a nucleic acid    molecule of (a) to (c) under under stringent hybridisation    conditions and conferring an increase in the amount of the fine    chemical in an organism or a part thereof;-   f) nucleic acid molecule encoding a polypeptide, the polypeptide    being derived by substituting, deleting and/or adding one or more    amino acids of the amino acid sequence of the polypeptide encoded by    the nucleic acid molecules (a) to (d), preferably to (a) to (c) and    conferring an increase in the amount of the fine chemical in an    organism or a part thereof;-   g) nucleic acid molecule encoding a fragment or an epitope of a    polypeptide which is encoded by one of the nucleic acid molecules    of (a) to (e), preferably to (a) to (c) and conferring an increase    in the amount of the fine chemical in an organism or a part thereof;-   h) nucleic acid molecule comprising a nucleic acid molecule which is    obtained by amplifying nucleic acid molecules from a cDNA library or    a genomic library using the primers as depicted in SEQ ID NO: 53 or    SEQ ID NO: 54 and conferring an increase in the amount of the fine    chemical in an organism or a part thereof;-   i) nucleic acid molecule encoding a polypeptide which is isolated,    e.g. from an expression library, with the aid of monoclonal    antibodies against a polypeptide encoded by one of the nucleic acid    molecules of (a) to (h), preferably to (a) to (c), and and    conferring an increase in the amount of the fine chemical in an    organism or a part thereof;-   j) nucleic acid molecule which encodes a polypeptide comprising the    consensus sequence as depicted in SEQ ID NO: 47, SEQ ID NO: 48, SEQ    ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:    397, SEQ ID NO: 398, SEQ ID NO: 399 and/or SEQ ID NO: 400 and    conferring an increase in the amount of the fine chemical in an    organism or a part thereof; and/or-   k) nucleic acid molecule which is obtainable by screening a suitable    library under stringent conditions with a probe comprising one of    the sequences of the nucleic acid molecule of (a) to a), preferably    to (a) to (c), or with a fragment of at least 15 nt, preferably 20    nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid    molecule characterized in (a) to (j), preferably to (a) to (c), and    conferring an increase in the amount of the fine chemical in an    organism or a part thereof;    or which comprises a sequence which is complementary thereto.

In one embodiment, the nucleic acid molecule used in the processdistinguishes over the sequence as depicted in SEQ ID NO: 1, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391 or 393 by one or more nucleotides ordoes not consist of the sequence as depicted in SEQ ID NO: 1, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179,181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291,293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319,321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,377, 379, 381, 383, 385, 387, 389, 391 or 393. In one embodiment, thenucleic acid molecule of the present invention is less than 100%,99.999%, 99.99%, 99.9% or 99% identical to the sequence as depicted inSEQ ID NO: 1, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197,199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225,227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253,255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281,283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309,311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337,339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365,367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393.In another embodiment, the nucleic acid molecule does not encode apolypeptide of the sequence as depicted in SEQ ID NO: 2, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392 or 394. In another embodiment, thenucleic acid molecule does not encode a polypeptide of the sequence ofthe present invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%identical to the sequence as depicted in SEQ ID NO: 2, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392 or 394. In another embodiment, thenucleic acid molecule encodes a polypeptide of the sequence as depictedin SEQ ID NO: 2, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394and distinguishes over said sequence by one or more amino acidspreferably by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In furtheranother advantageously embodiment, the nucleic acid molecule used in theprocess distinguishes over the sequence as depicted in SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 SEQ IDNO: 43 or SEQ ID NO: 45 by one or more nucleotides or does not consistof the sequence as depicted in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 SEQ ID NO: 43 or SEQ ID NO: 45.In one advantageously embodiment, the nucleic acid molecule of thepresent invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%identical to the sequence as depicted in SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 SEQ ID NO: 43 or SEQ IDNO: 45. In another advantageously embodiment, the nucleic acid moleculedoes not encode a polypeptide of the sequence as depicted in SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ IDNO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,SEQ ID NO: 44 or SEQ ID NO: 46.

Unless otherwise specified, the terms “polynucleotides”, “nucleic acid”and “nucleic acid molecule” are interchangeably in the present context.Unless otherwise specified, the terms “peptide”, “polypeptide” and“protein” are interchangeably in the present context. The term“sequence” may relate to polynucleotides, nucleic acids, nucleic acidmolecules, peptides, polypeptides and proteins, depending on the contextin which the term “sequence” is used. The terms “gene(s)”,“polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid molecule(s)” as used herein refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. The terms refer only to the primary structure ofthe molecule.

Thus, the terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”,“nucleotide sequence”, or “nucleic acid molecule(s)” as used hereininclude double- and single-stranded DNA and RNA. They also include knowntypes of modifications, for example, methylation, “caps”, substitutionsof one or more of the naturally occurring nucleotides with an analog.Preferably, the DNA or RNA sequence of the invention comprises a codingsequence encoding the herein defined polypeptide.

A “coding sequence” is a nucleotide sequence, which is transcribed intomRNA and/or translated into a polypeptide when placed under the controlof appropriate regulatory sequences. The boundaries of the codingsequence are determined by a translation start codon at the 5′-terminusand a translation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to mRNA, cDNA, recombinant nucleotidesequences or genomic DNA, while introns may be present as well undercertain circumstances.

Nucleic acid molecules with the sequence as depicted in SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQID NO: 41 SEQ ID NO: 43 or SEQ ID NO: 45, nucleic acid molecules whichare derived from the amino acid sequences as depicted in SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46 or from polypeptidescomprising the consensus sequence as depicted in SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399 and/or SEQ ID NO: 400, ortheir derivatives or homologues encoding polypeptides with the enzymaticor biological activity of a protein as depicted in SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32,SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO: 44 or SEQ ID NO: 46 and/or conferring a fine chemicalincrease after increasing its expression or activity are advantageouslyincreased in the process according to the invention.

In one embodiment, said sequences are cloned into nucleic acidconstructs, either individually or in combination. These nucleic acidconstructs enable an optimal synthesis of the fine chemical produced inthe process according to the invention.

Nucleic acid molecules, which are advantageous for the process accordingto the invention and which encode polypeptides with the biologicalactivity of the protein of the invention can be determined fromgenerally accessible databases.

Those, which must be mentioned, in particular in this context aregeneral gene databases such as the EMBL database (Stoesser G. et al.,Nucleic Acids Res 2001, Vol. 29, 17-21), the GenBank database (Benson D.A. et al., Nucleic Acids Res 2000, Vol. 28, 15-18), or the PIR database(Barker W. C. et al., Nucleic Acids Res. 1999, Vol. 27, 39-43). It isfurthermore possible to use organism-specific gene databases fordetermining advantageous sequences, in the case of yeast for exampleadvantageously the SGD database (Cherry J. M. et al., Nucleic Acids Res.1998, Vol. 26, 73-80) or the MIPS database (Mewes H. W. et al., NucleicAcids Res. 1999, Vol. 27, 44-48), in the case of E. coli the GenProtECdatabase (http://web.bham.ac.uk/bcm4ght6/res.html), and in the case ofArabidopsis the TAIR-database (Huala, E. et al., Nucleic Acids Res. 2001Vol. 29(1), 102-5) or the MIPS database.

The nucleic acid molecules used in the process according to theinvention take the form of isolated nucleic acid sequences, which encodepolypeptides having the biological activity represented by a protein asdepicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300,302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328,330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,386, 388, 390, 392 or 394 and conferring the fine chemical increase.

The nucleic acid sequence(s) used in the process for the production ofthe fine chemical in transgenic organisms originate advantageously froman eukaryote but may also originate from a prokaryote or anarchebacterium, thus it can derived from e.g. a microorganism, an animalor a plant.

For the purposes of the invention, as a rule the plural is intended toencompass the singular and vice versa.

In order to improve the introduction of the nucleic acid sequences andthe expression of the sequences in the transgenic organisms, which areused in the process, the nucleic acid sequences are incorporated into anucleic acid construct and/or a vector. In addition to the hereindescribed sequences which are used in the process according to theinvention, further nucleic acid sequences, advantageously ofbiosynthesis genes of amino acids, carbohydrates, lipids, fatty acids,vitamins etc. produced in the process according to the invention, mayadditionally be present in the nucleic acid construct or in the vectorand may be introduced into the organism together. However, theseadditional sequences may also be introduced into the organisms viaother, separate nucleic acid constructs or vectors.

Using the herein mentioned cloning vectors and transformation methodssuch as those which are published and cited in: Plant Molecular Biologyand Biotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7, pp. 71-119(1993); F. F. White, Vectors for Gene Transfer in Higher Plants; in:Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung and R.Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, vol. 1, Engineering and Utilization,Ed.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu.Rev. Plant. Physiol. Plant Molec. Biol. 42 (1991), 205-225)) and furthercited below, the nucleic acids may be used for the recombinantmodification of a wide range of organisms, in particular prokaryotic oreukaryotic microorganisms or plants, so that they become a better andmore efficient producer of the fine chemical produced in the processaccording to the invention. This improved production, or productionefficiency, of the fine chemical or products derived there from, such asmodified proteins, can be brought about by a direct effect of themanipulation or by an indirect effect of this manipulation.

In one embodiment, the nucleic acid molecule according to the inventionoriginates from a plant, such as a plant selected from the familiesAceraceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae,Cucurbitaceae, Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae,Papaveraceae, Rosaceae, Salicaceae, Solanaceae, Arecaceae, Bromeliaceae,Cyperaceae, lridaceae, Liliaceae, Orchidaceae, Gentianaceae, Labiaceae,Magnoliaceae, Ranunculaceae, Carifolaceae, Rubiaceae, Scrophulariaceae,Caryophyllaceae, Ericaceae, Polygonaceae, Violaceae, Juncaceae orPoaceae and preferably from a plant selected from the group of thefamilies Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae,Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae. Preferred arecrop plants and in particular plants mentioned herein above as hostplants such as the families and genera mentioned above for examplepreferred the species Anacardium occidentale, Calendula officinalis,Carthamus tinctorius, Cichorium intybus, Cynara scolymus, Helianthusannus, Tagetes lucida, Tagetes erecta, Tagetes tenuifolia; Daucuscarota; Corylus avellana, Corylus colurna, Borago officinalis; Brassicanapus, Brassica rapa ssp., Sinapis arvensis, Brassica juncea, Brassicajuncea var. juncea, Brassica juncea var. crispifolia, Brassica junceavar. foliosa, Brassica nigra, Brassica sinapioides, Melanosinapiscommunis, Brassica oleracea, Arabidopsis thaliana, Anana comosus, Ananasananas, Bromelia comosa, Carica papaya, Cannabis sative, Ipomoeabatatus, Ipomoea pandurata, Convolvulus batatas, Convolvulus tiliaceus,Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba, Convolvuluspanduratus, Beta vulgaris, Beta vulgaris var. altissima, Beta vulgarisvar. vulgaris, Beta maritima, Beta vulgaris var. perennis, Beta vulgarisvar. conditiva, Beta vulgaris var. esculenta, Cucurbita maxima,Cucurbita mixta, Cucurbita pepo, Cucurbita moschata, Olea europaea,Manihot utilissima, Janipha manihot, Jatropha manihot, Manihot aipil,Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta,Ricinus communis, Pisum sativum, Pisum arvense, Pisum humile, Medicagosativa, Medicago falcata, Medicago varia, Glycine max Dolichos soja,Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida, Sojamax, Cocos nucifera, Pelargonium grossularioides, Oleum cocoas, Laurusnobilis, Persea americana, Arachis hypogaea, Linum usitatissimum, Linumhumile, Linum austriacum, Linum bienne, Linum angustifolium, Linumcatharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum,Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var.lewisii, Linum pratense, Linum trigynum, Punica granatum, Gossypiumhirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum,Gossypium thurberi, Musa nana, Musa acuminata, Musa paradisiaca, Musaspp., Elaeis guineensis, Papaver orientale, Papaver rhoeas, Papaverdubium, Sesamum indicum, Piper aduncum, Piper amalago, Piperangustifolium, Piper auritum, Piper betel, Piper cubeba, Piper longum,Piper nigrum, Piper retrofractum, Artanthe adunca, Artanthe elongata,Peperomia elongata, Piper elongatum, Steffensia elongata, Hordeumvulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeumdistichon, Hordeum aegiceras, Hordeum hexastichon, Hordeum hexastichum,Hordeum irregulare, Hordeum sativum, Hordeum secalinum, Avena sativa,Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida,Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghumvulgare, Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghumaethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum,Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghumsubglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcushalepensis, Sorghum miliaceum millet, Panicum militaceum, Zea mays,Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum,Triticum macha, Triticum sativum or Triticum vulgare, Cofea spp., Coffeaarabica, Coffea canephora, Coffea liberica, Capsicum annuum, Capsicumannuum var. glabriusculum, Capsicum frutescens, Capsicum annuum,Nicotiana tabacum, Solanum tuberosum, Solanum melongena, Lycopersiconesculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme, Solanumintegrifolium, Solanum lycopersicum Theobroma cacao or Camelliasinensis.

In one embodiment, the nucleic acid molecule sequence originatesadvantageously from a microorganism as mentioned above under hostorganism such as a fungus for example the genera Aspergillus,Penicillium or Claviceps or from yeasts such as the genera Pichia,Torulopsis, Hansenula, Schizosaccharomyces, Candida, Rhodotorula orSaccharomyces, very especially advantageously from the yeast of thefamily Saccharomycetaceae, such as the advantageous genus Saccharomycesand the very advantageous genus and species Saccharomyces cerevisiae forthe production of the fine chemical in microorganims.

The skilled worker knows other suitable sources for the production offine chemicals, which present also useful nucleic acid molecule sources.They include in general all prokaryotic or eukaryotic cells, preferablyunicellular microorganisms, such as fungi like the genus Claviceps orAspergillus or gram-positive bacteria such as the genera Bacillus,Corynebacterium, Micrococcus, Brevibacterium, Rhodococcus, Nocardia,Caseobacter or Arthrobacter or gram-negative bacteria such as the generaEscherichia, Flavobacterium or Salmonella, or yeasts such as the generaRhodotorula, Hansenula or Candida.

Production strains which are especially advantageously selected in theprocess according to the invention are microorganisms selected from thegroup of the families Actinomycetaceae, Bacillaceae, Brevibacteriaceae,Corynebacteriaceae, Enterobacteriacae, Gordoniaceae, Micrococcaceae,Mycobacteriaceae, Nocardiaceae, Pseudomonaceae, Rhizobiaceae,Streptomycetaceae, Chaetomiaceae, Choanephoraceae, Cryptococcaceae,Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae,Mucoraceae, Pythiaceae, Saccharomycetaceae, Saprolegniaceae,Schizosaccharomycetaceae, Sodariaceae, Sporobolomycetaceae,Tuberculariaceae, Adelotheciaceae, Dinophyceae, Ditrichaceae andPrasinophyceaeor of the genera and species consisting of Hansenulaanomala, Candida utilis, Claviceps purpurea, Bacillus circulans,Bacillus subtilis, Bacillus sp., Brevibacterium albidum, Brevibacteriumalbum, Brevibacterium cerinum, Brevibacterium flavum, Brevibacteriumglutamigenes, Brevibacterium iodinum, Brevibacterium ketoglutamicum,Brevibacterium lactofermentum, Brevibacterium linens, Brevibacteriumroseum, Brevibacterium saccharolyticum, Brevibacterium sp.,Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum,Corynebacterium ammoniagenes, Corynebacterium glutamicum (=Micrococcusglutamicum), Corynebacterium melassecola, Corynebacterium sp. orEscherichia coli, specifically Escherichia coli K12 and its describedstrains.

However, it is also possible to use artificial sequences, which differin one or more bases (=nucleotides) from the nucleic acid sequencesfound in organisms, or in one or more amino acid molecules frompolypeptide sequences found in organisms, in particular from thepolypeptide sequences as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392 or 394 or the functionalhomologues thereof as described herein, preferably conferringabove-mentioned biological activity, i.e. conferring the fine chemicalincrease after increasing its activity, e.g. having the biologicalactivity represented by a protein as depicted in SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370,372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394.

In the process according to the invention nucleic acid sequences can beused, which, if appropriate, contain synthetic, non-natural or modifiednucleotide bases, which can be incorporated into DNA or RNA. Saidsynthetic, non-natural or modified bases can for example increase thestability of the nucleic acid molecule outside or inside a cell. Thenucleic acid molecules of the invention can contain the samemodifications as aforementioned.

As used in the present context the term “nucleic acid molecule” may alsoencompass the untranslated sequence located at the 3′ and at the 5′ endof the coding gene region, for example at least 500, preferably 200,especially preferably 100, nucleotides of the sequence upstream of the5′ end of the coding region and at least 100, preferably 50, especiallypreferably 20, nucleotides of the sequence downstream of the 3′ end ofthe coding gene region. It is often advantageous only to choose thecoding region for cloning and expression purposes.

Preferably, the nucleic acid molecule used in the process according tothe invention or the nucleic acid molecule of the invention is anisolated nucleic acid molecule.

An “isolated” polynucleotide or nucleic acid molecule is separated fromother polynucleotides or nucleic acid molecules, which are present inthe natural source of the nucleic acid molecule. An isolated nucleicacid molecule may be a chromosomal fragment of several kb, orpreferably, a molecule only comprising the coding region of the gene.Accordingly, an isolated nucleic acid molecule of the invention maycomprise chromosomal regions, which are adjacent 5′ and 3′ or furtheradjacent chromosomal regions, but preferably comprises no such sequenceswhich naturally flank the nucleic acid molecule sequence in the genomicor chromosomal context in the organism from which the nucleic acidmolecule originates (for example sequences which are adjacent to theregions encoding the 5′- and 3′-UTRs of the nucleic acid molecule). Invarious embodiments, the isolated nucleic acid molecule used in theprocess according to the invention may, for example comprise less thanapproximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb nucleotidesequences which naturally flank the nucleic acid molecule in the genomicDNA of the cell from which the nucleic acid molecule originates.

The nucleic acid molecules used in the process, for example thepolynucleotides of the invention or of a part thereof can be isolatedusing molecular-biological standard techniques and the sequenceinformation provided herein. Also, for example a homologous sequence orhomologous, conserved sequence regions at the DNA or amino acid levelcan be identified with the aid of comparison algorithms. The former canbe used as hybridization probes under standard hybridization techniques(for example those described in Sambrook et al., Molecular Cloning: ALaboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) for isolatingfurther nucleic acid sequences useful in this process.

A nucleic acid molecule encompassing a complete sequence of the nucleicacid molecules used in the process, for example the polynucleotide ofthe invention, or a part thereof may additionally be isolated bypolymerase chain reaction, oligonucleotide primers based on thissequence or on parts thereof being used. For example, a nucleic acidmolecule comprising the complete sequence or part thereof can beisolated by polymerase chain reaction using oligonucleotide primerswhich have been generated on the basis of this sequence for example,mRNA can be isolated from cells (for example by means of the guanidiniumthiocyanate extraction method of Chirgwin et al. (1979) Biochemistry18:5294-5299) and cDNA can be generated by means of reversetranscriptase (for example Moloney MLV reverse transcriptase, availablefrom Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, obtainablefrom Seikagaku America, Inc., St. Petersburg, Fla.).

Synthetic oligonucleotide primers for the amplification, e.g. as shownin SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 395 or SEQ ID NO: 396, bymeans of polymerase chain reaction can be generated on the basis of asequence shown herein, for example the sequence as depicted in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279,281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363,365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or393.

Moreover, it is possible to identify conserved regions from variousorganisms by carrying out protein sequence alignments with thepolypeptide used in the process of the invention, in particular withsequences of the polypeptide of the invention, from which conservedregions, and in turn, degenerate primers can be derived. Conservedregions are those, which show a very little variation in the amino acidin one particular position of several homologs from different origin.The consensus sequences as depicted in SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 397,SEQ ID NO: 398, SEQ ID NO: 399 and/or SEQ ID NO: 400 are derived fromsaid alignments.

Degenerated primers can then be utilized by PCR for the amplification offragments of novel proteins having above-mentioned activity, e.g.conferring the increase of the fine chemical after increasing theexpression or activity or having the biological activity represented bya protein as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392 or 394 or further functional homologs ofthe polypeptide of the invention from other organisms.

These fragments can then be utilized as hybridization probe forisolating the complete gene sequence. As an alternative, the missing 5′and 3′ sequences can be isolated by means of RACE-PCR (rapidamplification of cDNA ends). A nucleic acid molecule according to theinvention can be amplified using cDNA or, as an alternative, genomic DNAas template and suitable oligonucleotide primers, following standard PCRamplification techniques. The nucleic acid molecule amplified thus canbe cloned into a suitable vector and characterized by means of DNAsequence analysis. Oligonucleotides, which correspond to one of thenucleic acid molecules used in the process can be generated by standardsynthesis methods, for example using an automatic DNA synthesizer.

Nucleic acid molecules which are advantageously for the processaccording to the invention can be isolated based on their homology tothe nucleic acid molecules disclosed herein using the sequences or partthereof as hybridization probe and following standard hybridizationtechniques under stringent hybridization conditions. In this context, itis possible to use, for example, isolated nucleic acid molecules of atleast 15, 20, 25, 30, 35, 40, 50, 60 or more nucleotides, preferably ofat least 15, 20 or 25 nucleotides in length which hybridize understringent conditions with the above-described nucleic acid molecules, inparticular with those which encompass a nucleotide sequence of thenucleic acid molecule used in the process of the invention or encoding aprotein used in the invention or of the nucleic acid molecule of theinvention. Nucleic acid molecules with 30, 50, 100, 250 or morenucleotides may also be used.

The term “homology” means that the respective nucleic acid molecules orencoded proteins are functionally and/or structurally equivalent. Thenucleic acid molecules that are homologous to the nucleic acid moleculesdescribed above and that are derivatives of said nucleic acid moleculesare, for example, variations of said nucleic acid molecules whichrepresent modifications having the same biological function, inparticular encoding proteins with the same or substantially the samebiological function. They may be naturally occurring variations, such assequences from other plant varieties or species, or mutations. Thesemutations may occur naturally or may be obtained by mutagenesistechniques. The allelic variations may be naturally occurring allelicvariants as well as synthetically produced or genetically engineeredvariants. Structurally equivalents can, for example, be identified bytesting the binding of said polypeptide to antibodies or computer basedpredictions. Structurally equivalent have the similar immunologicalcharacteristic, e.g. comprise similar epitopes.

By “hybridizing” it is meant that such nucleic acid molecules hybridizeunder conventional hybridization conditions, preferably under stringentconditions such as described by, e.g., Sambrook (Molecular Cloning; ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989)) or in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

According to the invention, DNA as well as RNA molecules of the nucleicacid of the invention can be used as probes. Further, as template forthe identification of functional homologues Northern blot assays as wellas Southern blot assays can be performed. The Northern blot assayadvantageously provides further informations about the expressed geneproduct: e.g. expression pattern, occurance of processing steps, likesplicing and capping, etc. The Southern blot assay provides additionalinformation about the chromosomal localization and organization of thegene encoding the nucleic acid molecule of the invention.

A preferred, nonlimiting example of stringent hydridization conditionsare hybridizations in 6× sodium chloride/sodium citrate (=SSC) atapproximately 45° C., followed by one or more wash steps in 0.2×SSC,0.1% SDS at 50 to 65° C., for example at 50° C., 55° C. or 60° C. Theskilled worker knows that these hybridization conditions differ as afunction of the type of the nucleic acid and, for example when organicsolvents are present, with regard to the temperature and concentrationof the buffer. The temperature under “standard hybridization conditions”differs for example as a function of the type of the nucleic acidbetween 42° C. and 58° C., preferably between 45° C. and 50° C. in anaqueous buffer with a concentration of 0.1×0.5×, 1×, 2×, 3×, 4× or 5×SSC(pH 7.2). If organic solvent(s) is/are present in the abovementionedbuffer, for example 50% formamide, the temperature under standardconditions is approximately 40° C., 42° C. or 45° C. The hybridizationconditions for DNA:DNA hybrids are preferably for example 0.1×SSC and20° C., 25° C., 30° C., 35° C., 40° C. or 45° C., preferably between 30°C. and 45° C. The hybridization conditions for DNA:RNA hybrids arepreferably for example 0.1×SSC and 30° C., 35° C., 40° C., 45° C., 50°C. or 55° C., preferably between 45° C. and 55° C. The abovementionedhybridization temperatures are determined for example for a nucleic acidapproximately 100 bp (=base pairs) in length and a G+C content of 50% inthe absence of formamide. The skilled worker knows to determine thehybridization conditions required with the aid of textbooks, for examplethe ones mentioned above, or from the following textbooks: Sambrook etal., “Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames andHiggins (Ed.) 1985, “Nucleic Acids Hybridization: A Practical Approach”,IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991,“Essential Molecular Biology: A Practical Approach”, IRL Press at OxfordUniversity Press, Oxford.

A further example of one such stringent hybridization condition ishybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at65° C. for one hour. Alternatively, an exemplary stringent hybridizationcondition is in 50% formamide, 4×SSC at 42° C. Further, the conditionsduring the wash step can be selected from the range of conditionsdelimited by low-stringency conditions (approximately 2×SSC at 50° C.)and high-stringency conditions (approximately 0.2×SSC at 50° C.,preferably at 65° C.) (20×SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). Inaddition, the temperature during the wash step can be raised fromlow-stringency conditions at room temperature, approximately 22° C., tohigher-stringency conditions at approximately 65° C. Both of theparameters salt concentration and temperature can be variedsimultaneously, or else one of the two parameters can be kept constantwhile only the other is varied. Denaturants, for example formamide orSDS, may also be employed during the hybridization. In the presence of50% formamide, hybridization is preferably effected at 42° C. Relevantfactors like i) length of treatment, ii) salt conditions, iii) detergentconditions, iv) competitor DNAs, v) temperature and vi) probe selectioncan be combined case by case so that not all possibilities can bementioned herein.

Thus, in a preferred embodiment, Northern blots are prehybridized withRothi-Hybri-Quick buffer (Roth, Karlsruhe) at 68° C. for 2 h.Hybridzation with radioactive labelled probe is done overnight at 68° C.Subsequent washing steps are performed at 68° C. with 1×SSC.

For Southern blot assays the membrane is prehybridized withRothi-Hybri-Quick buffer (Roth, Karlsruhe) at 68° C. for 2 h. Thehybridzation with radioactive labelled probe is conducted over night at68° C. Subsequently the hybridization buffer is discarded and the filtershortly washed using 2×SSC; 0.1% SDS. After discarding the washingbuffer new 2×SSC; 0.1% SDS buffer is added and incubated at 68° C. for15 minutes. This washing step is performed twice followed by anadditional washing step using 1×SSC; 0.1% SDS at 68° C. for 10 min.

Some further examples of conditions for DNA hybridization (Southem blotassays) and wash step are shown hereinbelow:

-   (1) Hybridization conditions can be selected, for example, from the    following conditions:-   a) 4×SSC at 65° C.,-   b) 6×SSC at 45° C.,-   c) 6×SSC, 100 mg/ml denatured fragmented fish sperm DNA at 68° C.,-   d) 6×SSC, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA at 68° C.,-   e) 6×SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon sperm DNA,    50% formamide at 42° C.,-   f) 50% formamide, 4×SSC at 42° C.,-   g) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll,    0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750    mM NaCl, 75 mM sodium citrate at 42° C.,-   h) 2× or 4×SSC at 50° C. (low-stringency condition), or-   i) 30 to 40% formamide, 2× or 4×SSC at 42° C. (low-stringency    condition).-   (2) Wash steps can be selected, for example, from the following    conditions:-   a) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.-   b) 0.1×SSC at 65° C.-   c) 0.1×SSC, 0.5% SDS at 68° C.-   d) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C.-   e) 0.2×SSC, 0.1% SDS at 42° C.-   f) 2×SSC at 65° C. (low-stringency condition).

Polypeptides having above-mentioned biological activity, i.e. conferringthe fine chemical increase, derived from other organisms, can be encodedby other DNA sequences which hybridize to the sequences shown in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279,281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363,365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or393 under relaxed hybridization conditions and which code on expressionfor peptides having the fine chemical increasing activity.

Further, some applications have to be performed at low stringencyhybridisation conditions, without any consequences for the specificityof the hybridisation. For example, a Southern blot analysis of total DNAcould be probed with a nucleic acid molecule of the present inventionand washed at low stringency (55° C. in 2×SSPE0, 1% SDS). Thehybridisation analysis could reveal a simple pattern of only genesencoding polypeptides of the present invention or used in the process ofthe invention, e.g. having herein-mentioned activity of increasing thefine chemical. A further example of such low-stringent hybridizationconditions is 4×SSC at 50° C. or hybridization with 30 to 40% formamideat 42° C. Such molecules comprise those which are fragments, analoguesor derivatives of the polypeptide of the invention or used in theprocess of the invention and differ, for example, by way of amino acidand/or nucleotide deletion(s), insertion(s), substitution (s),addition(s) and/or recombination (s) or any other modification(s) knownin the art either alone or in combination from the above-described aminoacid sequences or their underlying nucleotide sequence(s). However, itis preferred to use high stringency hybridisation conditions.

Hybridization should advantageously be carried out with fragments of atleast 5, 10, 15, 20, 25, 30, 35 or 40 bp, advantageously at least 50,60, 70 or 80 bp, preferably at least 90, 100 or 110 bp. Most preferablyare fragments of at least 15, 20, 25 or 30 bp. Preferably are alsohybridizations with at least 100 bp or 200, very especially preferablyat least 400 bp in length. In an especially preferred embodiment, thehybridization should be carried out with the entire nucleic acidsequence with conditions described above.

The terms “fragment”, “fragment of a sequence” or “part of a sequence”mean a truncated sequence of the original sequence referred to. Thetruncated sequence (nucleic acid or protein sequence) can vary widely inlength; the minimum size being a sequence of sufficient size to providea sequence with at least a comparable function and/or biologicalactivity of the original sequence referred to or hybidizing with thenucleic acid molecule of the invention or used in the process of theinvention under stringend conditions, while the maximum size is notcritical. In some applications, the maximum size usually is notsubstantially greater than that required to provide the desired activityand/or function(s) of the original sequence.

Typically, the truncated amino acid sequence will range from about 5 toabout 260 amino acids in length. More typically, however, the sequencewill be a maximum of about 220 amino acids in length, preferably amaximum of about 215 or 100 amino acids. It is usually desirable toselect sequences of at least about 100, 120 or 150 amino acids, up to amaximum of about 200 or 250 amino acids.

The term “epitope” relates to specific immunoreactive sites within anantigen, also known as antigenic determinates. These epitopes can be alinear array of monomers in a polymeric composition—such as amino acidsin a protein—or consist of or comprise a more complex secondary ortertiary structure. Those of skill will recognize that immunogens (i.e.,substances capable of eliciting an immune response) are antigens;however, some antigen, such as haptens, are not immunogens but may bemade immunogenic by coupling to a carrier molecule. The term “antigen”includes references to a substance to which an antibody can be generatedand/or to which the antibody is specifically immunoreactive.

In one embodiment the present invention relates to a epitope of thepolypeptide of the present invention or used in the process of thepresent invention and conferring above mentioned biological activity,preferably conferring an increase in the fine chemical.

The term “one or several amino acids” relates to at least one amino acidbut not more than that number of amino acids, which would result in ahomology of below 50% identity. Preferably, the identity is more than70% or 80%, more preferred are 85%, 90%, 91%, 92%, 93%, 94% or 95%, evenmore preferred are 96%, 97%, 98%, or 99% identity.

Further, the nucleic acid molecule of the invention comprises a nucleicacid molecule, which is a complement of one of the nucleotide sequencesof above mentioned nucleic acid molecules or a portion thereof. Anucleic acid molecule which is complementary to one of the nucleotidesequences as depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295,297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351,353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379,381, 383, 385, 387, 389, 391 or 393 is one which is sufficientlycomplementary to one of the nucleotide sequences as depicted in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279,281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363,365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or393 such that it can hybridize to one of the nucleotide sequences asdepicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327,329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,385, 387, 389, 391 or 393, thereby forming a stable duplex. Preferably,the hybridisation is performed under stringent hybrization conditions.However, a complement of one of the herein disclosed sequences ispreferably a sequence complement thereto according to the base pairingof nucleic acid molecules well known to the skilled person. For example,the bases A and G undergo base pairing with the bases T and U or C,resp. and visa versa. Modifications of the bases can influence thebase-pairing partner.

The nucleic acid molecule of the invention comprises a nucleotidesequence which is at least about 30%, 35%, 40% or 45%, preferably atleast about 50%, 55%, 60% or 65%, more preferably at least about 70%,80%, or 90%, and even more preferably at least about 95%, 97%, 98%, 99%most preferably at least about 99.1%; 99.2%, 99.3%; 99.4%; 99.5%; 99.6%;99.7%; 99.8% or 99.9% or more homo-logous to a nucleotide sequence asdepicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327,329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,385, 387, 389, 391 or 393, or a portion thereof and preferably has abovementioned biological activity, in particular having a fine chemicalincreasing activity after increasing the biological acitivity of aprotein having the biological activity represented by a protein asdepicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258., 260, 262, 264, 266, 268, 270, 272,274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300,302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328,330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,386, 388, 390, 392 or 394 and their respective gene products.

The nucleic acid molecule of the invention comprises a nucleotidesequence which hybridizes, preferably hybridizes under stringentconditions as defined herein, to one of the nucleotide sequences asdepicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327,329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,385, 387, 389, 391 or 393, or a portion thereof and encodes a proteinhaving above-mentioned biological activity, e.g. conferring the finechemical increase, and optionally, having the biological activity ofYNL090W.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393, forexample a fragment which can be used as a probe or primer or a fragmentencoding a biologically active portion of the polypeptide of the presentinvention or of a polypeptide used in the process of the presentinvention, i.e. having above-mentioned activity, e.g. conferring anincrease of methionine if its activity is increased. The nucleotidesequences determined from the cloning of the present protein accordingto the invention encoding gene allows for the generation of probes andprimers designed for use in identifying and/or cloning its homologues inother cell types and organisms. The probe/primer typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 15 preferably about 20 or 25,more preferably about 40, 50 or 75 consecutive nucleotides of a sensestrand of one of the sequences set forth, e.g., in SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393, ananti-sense sequence of one of the sequences, e.g., set forth in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279,281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363,365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or393, or naturally occurring mutants thereof. Primers based on anucleotide of invention can be used in PCR reactions to clone homologuesof the polypeptide of the invention or of the polypeptide used in theprocess of the invention, e.g. as the primers described in the examplesof the present invention, e.g. as shown in the examples. A PCR with theprimers shown in SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 395 or SEQ IDNO: 396 will at least result in a fragment of gene products as depictedin the sequences SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269,271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325,327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353,355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,383, 385, 387, 389, 391 or 393.

Primer sets are interchangable. The person skilled in the art knows tocombine said primers to result in the desired product, e.g. in afull-length clone or a partial sequence. Probes based on the sequencesof the nucleic acid molecule of the invention or used in the process ofthe present invention can be used to detect transcripts or genomicsequences encoding the same or homologous proteins. The probe canfurther comprise a label group attached thereto, e.g. the label groupcan be a radioisotope, a fluorescent compound, an enzyme, or an enzymeco-factor. Such probes can be used as a part of a genomic marker testkit for identifying cells which express an polypepetide of the inventionor used in the process of the present invention, such as by measuring alevel of an encoding nucleic acid molecule in a sample of cells, e.g.,detecting mRNA levels or determining, whether a genomic gene comprisingthe sequence of the polynucleotide of the invention or used in theprocesss of the present invention has been mutated or deleted.

The nucleic acid molecule of the invention encodes a polypeptide orportion thereof which includes an amino acid sequence which issufficiently homologous to the amino acid sequence as depicted in SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308,310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or394 such that the protein or portion thereof maintains the ability toparticipate in the fine chemical production, in particular an amino acidincreasing activity as mentioned above or as described in the examplesin plants or microorganisms is comprised.

As used herein, the language “sufficiently homologous” refers toproteins or portions thereof which have amino acid sequences whichinclude a minimum number of identical or equivalent amino acid residues(e.g., an amino acid residue which has a similar side chain as an aminoacid residue in one of the sequences of the polypeptide of the presentinvention) to an amino acid sequence as depicted in SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340,342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 suchthat the protein or portion thereof is able to participate in theincrease of the fine chemical production. For examples having thebiological activity represented by a protein as depicted in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394are described herein.

In one embodiment, the nucleic acid molecule of the present inventioncomprises a nucleic acid that encodes a portion of the protein of thepresent invention. The protein is at least about 30%, 35%, 40%, 45% or50%, preferably at least about 55%, 60%, 65% or 70%, and more preferablyat least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and mostpreferably at least about 95%, 97%, 98%, 99% or more homologous to anentire amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392 or 394 and having above-mentionedactivity, e.g. conferring preferably the increase of the fine chemical.

Portions of proteins encoded by the nucleic acid molecule of theinvention are preferably biologically active, preferably havingabove-mentioned annotated activity, e.g. conferring an increase of thefine chemical after increase of activity.

As mentioned herein, the term “biologically active portion” is intendedto include a portion, e.g., a domain/motif, that confers increase of thefine chemical or has an immunological activity such that it is binds toan antibody binding specifially to the polypeptide of the presentinvention or a polypeptide used in the process of the present inventionfor producing the fine chemical;

The invention further relates to nucleic acid molecules that differ fromone of the nucleotide sequences as depicted in SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259,261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287,289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315,317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343,345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371,373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393 (and portionsthereof) due to degeneracy of the genetic code and thus encode apolypeptide of the present invention, in particular a polypeptide havingabove mentioned activity, e.g. conferring an increase in the finechemical in a organism. Advantageously, the nucleic acid molecule of theinvention comprises, or in an other embodiment has, a nucleotidesequence encoding a protein comprising, or in an other embodimenthaving, an amino acid sequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344,346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372,374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 or thefunctional homologues. In a still further embodiment, the nucleic acidmolecule of the invention encodes a full length protein which issubstantially homologous to an amino acid sequence as depicted in SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308,310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or394 or the functional homologues. However, in a preferred embodiment,the nucleic acid molecule of the present invention does not consist ofthe sequence as depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391 or 393.

In addition, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesmay exist within a population. Such genetic polymorphism in the geneencoding the polypeptide of the invention or comprising the nucleic acidmolecule of the invention may exist among individuals within apopulation due to natural variation.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding the polypeptideof the invention or comprising the nucleic acid molecule of theinvention or encoding the polypeptide used in the process of the presentinvention, preferably from a crop plant or from a microorgansim usefulfor the production of fine chemicals, in particular for the productionof the fine chemical. Such natural variations can typically result in1-5% variance in the nucleotide sequence of the gene. Any and all suchnucleotide variations and resulting amino acid polymorphisms in genesencoding a polypeptide of the invention or comprising a the nucleic acidmolecule of the invention that are the result of natural variation andthat do not alter the functional activity as described are intended tobe within the scope of the invention.

Nucleic acid molecules corresponding to natural variants homologues of anucleic acid molecule of the invention, which can also be a cDNA, can beisolated based on their homology to the nucleic acid molecules disclosedherein using the nucleic acid molecule of the invention, or a portionthereof, as a hybridization probe according to standard hybridizationtechniques under stringent hybridization conditions.

Accordingly, in another embodiment, a nucleic acid molecule of theinvention is at least 15, 20, 25 or 30 nucleotides in length.Preferably, it hybridizes under stringent conditions to a nucleic acidmolecule comprising a nucleotide sequence of the nucleic acid moleculeof the present invention or used in the process of the presentinvention, e.g. comprising the sequence as depicted in SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393. Thenucleic acid molecule is preferably at least 20, 30, 50, 100, 250 ormore nucleotides in length.

The term “hybridizes under stringent conditions” is defined above. Inone embodiment, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 30%, 40%, 50% or 65% identical toeach other typically remain hybridized to each other. Preferably, theconditions are such that sequences at least about 70%, more preferablyat least about 75% or 80%, and even more preferably at least about 85%,90% or 95% or more identical to each other typically remain hybridizedto each other.

Preferably, nucleic acid molecule of the invention that hybridizes understringent conditions to a sequence as depicted in SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393 correspondsto a naturally-occurring nucleic acid molecule of the invention. As usedherein, a “naturally-occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein). Preferably, the nucleic acid moleculeencodes a natural protein having above-mentioned activity, e.g.conferring the fine chemical increase after increasing the expression oractivity thereof or the activity of a protein of the invention or usedin the process of the invention.

In addition to naturally-occurring variants of the sequences of thepolypeptide or nucleic acid molecule of the invention as well as of thepolypeptide or nucleic acid molecule used in the process of theinvention that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into anucleotide sequence of the nucleic acid molecule encoding thepolypeptide of the invention or used in the process of the presentinvention, thereby leading to changes in the amino acid sequence of theencoded polypeptide, without altering the functional ability of thepolypeptide, preferably not decreasing said activity.

For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in asequence of the nucleic acid molecule of the invention or used in theprocess of the invention, e.g. in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391 or 393.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of one without altering the activity of saidpolypeptide, whereas an “essential” amino acid residue is required foran activity as mentioned above, e.g. leading to an increase in the finechemical in an organism after an increase of activity of thepolypeptide. Other amino acid residues, however, (e.g., those that arenot conserved or only semi-conserved in the domain having said activity)may not be essential for activity and thus are likely to be amenable toalteration without altering said activity.

Further, a person skilled in the art knows that the codon usage betweenorganisms can differ. Therefore, he may adapt the codon usage in thenucleic acid molecule of the present invention to the usage of theorganism in which the polynuclestide or polypeptide is expressed.

Accordingly, the invention relates to nucleic acid molecules encoding apolypeptide having above-mentioned biological activity, e.g. conferringan increase in the the fine chemical in an organism or part thereof thatcontain changes in amino acid residues that are not essential for saidactivity. Such polypeptides differ in amino acid sequence from asequence contained in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392 or 394 yet retain said activity describedherein. The nucleic acid molecule can comprise a nucleotide sequenceencoding a polypeptide, wherein the polypeptide comprises an amino acidsequence at least about 50% identical to an amino acid sequence of SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306,308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334,336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362,364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,392 or 394 and is capable of participation in the increase of productionof the fine chemical after increasing its activity, e.g. its expression.Preferably, the protein encoded by the nucleic acid molecule is at leastabout 60% identical to the sequence in SEQ ID NO: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318,320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374,376, 378, 380, 382, 384, 386, 388, 390, 392 or 394, more preferably atleast about 70% identical to one of the sequences in SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340,342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394, evenmore preferably at least about 80%, 90%, 95% homologous to the sequencein SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394, and most preferably at least about 96%, 97%, 98%, or99% identical to the sequence in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392 or 394.

To determine the percentage homology (=identity) of two amino acidsequences or of two nucleic acid molecules, the sequences are writtenone underneath the other for an optimal comparison (for example gaps maybe inserted into the sequence of a protein or of a nucleic acid in orderto generate an optimal alignment with the other protein or the othernucleic acid).

The amino acid residues or nucleic acid molecules at the correspondingamino acid positions or nucleotide positions are then compared. If aposition in one sequence is occupied by the same amino acid residue orthe same nucleic acid molecule as the corresponding position in theother sequence, the molecules are homologous at this position (i.e.amino acid or nucleic acid “homology” as used in the present contextcorresponds to amino acid or nucleic acid “identity”. The percentagehomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e. % homology=number ofidentical positions/total number of positions×100). The terms “homology”and “identity” are thus to be considered as synonyms.

For the determination of the percentage homology (=identity) of two ormore amino acids or of two or more nucleotide sequences several computersoftware programs have been developed. The homology of two or moresequences can be calculated with for example the software fasta, whichpresently has been used in the version fasta 3 (W. R. Pearson and D. J.Lipman (1988), Improved Tools for Biological Sequence Comparison. PNAS85:2444-2448; W. R. Pearson (1990) Rapid and Sensitive SequenceComparison with FASTP and FASTA, Methods in Enzymology 183:63-98; W. R.Pearson and D. J. Lipman (1988) Improved Tools for Biological SequenceComparison. PNAS 85:2444-2448; W. R. Pearson (1990); Rapid and SensitiveSequence Comparison with FASTP and FASTA Methods in Enzymology183:63-98). Another useful program for the calculation of homologies ofdifferent sequences is the standard blast program, which is included inthe Biomax pedant software (Biomax, Munich, Federal Republic ofGermany). This leads unfortunately sometimes to suboptimal results sinceblast does not always include complete sequences of the subject and thequerry. Nevertheless as this program is very efficient it can be usedfor the comparison of a huge number of sequences. The following settingsare typically used for such a comparisons of sequences:

-p Program Name [String]; -d Database [String]; default=nr; -i QueryFile [File In]; default=stdin; -e Expectation value (E) [Real];default=10.0; -m alignment view options: 0=pairwise; 1=query-anchoredshowing identities; 2=query-anchored no identities; 3=flatquery-anchored, show identities; 4=flat query-anchored, no identities;5=query-anchored no identities and blunt ends; 6=flat query-anchored, noidentities and blunt ends; 7=XML Blast output; 8=tabular; 9 tabular withcomment lines [Integer]; default=0; -o BLAST report Output File [FileOut] Optional; default=stdout; -F Filter query sequence (DUST withblastn, SEG with others) [String]; default=T; -G Cost to open a gap(zero invokes default behavior) [Integer]; default=0; -E Cost to extenda gap (zero invokes default behavior) [Integer]; default=0; -X X dropoffvalue for gapped alignment (in bits) (zero invokes default behavior);blastn 30, megablast 20, tblastx 0, all others 15 [Integer]; default=0;-I Show GI's in deflines [T/F]; default=F; -q Penalty for a nucleotidemismatch (blastn only) [Integer]; default=−3; -r Reward for a nucleotidematch (blastn only) [Integer]; default=1; -v Number of databasesequences to show one-line descriptions for (V) [Integer]; default=500;-b Number of database sequence to show alignments for (B) [Integer];default=250; -f Threshold for extending hits, default if zero; blastp11, blastn 0, blastx 12, tblastn 13; tblastx 13, megablast 0 [Integer];default=0; -g Perform gapped alignment (not available with tblastx)[T/F]; default=T; -Q Query Genetic code to use [Integer]; default=1; -DDB Genetic code (for tblast[nx] only) [Integer]; default=1; -a Number ofprocessors to use [Integer]; default=1; -O SeqAlign file [File Out]Optional; -J Believe the query defline [T/F]; default=F; -M Matrix[String]; default=BLOSUM62; -W Word size, default if zero (blastn 11,megablast 28, all others 3) [Integer]; default=0; -z Effective length ofthe database (use zero for the real size) [Real]; default=0; -K Numberof best hits from a region to keep (off by default, if used a value of100 is recommended) [Integer]; default=0; -P 0 for multiple hit, 1 forsingle hit [Integer]; default=0; -Y Effective length of the search space(use zero for the real size) [Real]; default=0; -S Query strands tosearch against database (for blast[nx], and tblastx); 3 is both, 1 istop, 2 is bottom [Integer]; default=3; -T Produce HTML output [T/F];default=F; -I Restrict search of database to list of GI's [String]Optional; -U Use lower case filtering of FASTA sequence [T/F] Optional;default=F; -y X dropoff value for ungapped extensions in bits (0.0invokes default behavior); blastn 20, megablast 10, all others 7 [Real];default=0.0; -Z X dropoff value for final gapped alignment in bits (0.0invokes default behavior); blastn/megablast 50, tblastx 0, all others 25[Integer]; default=0; -R PSI-TBLASTN checkpoint file [File In] Optional;-n MegaBlast search [T/F]; default=F; -L Location on query sequence[String] Optional; -A Multiple Hits window size, default if zero(blastn/megablast 0, all others 40 [Integer]; default=0; -w Frame shiftpenalty (OOF algorithm for blastx) [Integer]; default=0; -t Length ofthe largest intron allowed in tblastn for linking HSPs (0 disableslinking) [Integer]; default=0.

Results of high quality are reached by using the algorithm of Needlemanand Wunsch or Smith and Waterman. Therefore programs based on saidalgorithms are preferred. Advantageously the comparisons of sequencescan be done with the program PileUp (J. Mol. Evolution., 25, 351-360,1987, Higgins etal., CABIOS, 5 1989: 151-153) or preferably with theprograms Gap and BestFit, which are respectively based on the algorithmsof Needleman and Wunsch [J. Mol. Biol. 48; 443-453 (1970)] and Smith andWaterman [Adv. Appl. Math. 2; 482-489 (1981)]. Both programs are part ofthe GCG software-package [Genetics Computer Group, 575 Science Drive,Madison, Wis., USA 53711 (1991); Altschul et al. (1997) Nucleic AcidsRes. 25:3389 et seq.]. Therefore preferably the calculations todetermine the perentages of sequence homology are done with the programGap over the whole range of the sequences. The following standardadjustments for the comparison of nucleic acid sequences were used: gapweight: 50, length weight: 3, average match: 10.000, average mismatch:0.000.

For example a sequence which has a 80% homology with sequence SEQ ID NO:1 at the nucleic acid level is understood as meaning a sequence which,upon comparison with the sequence SEQ ID NO: 1 by the above Gap programalgorithm with the above parameter set, has a 80% homology.

In the state of the art, homology between two polypeptides is alsounderstood as meaning the identity of the amino acid sequence over ineach case the entire sequence length which is calculated by comparisonwith the aid of the program algorithm GAP (Wisconsin Package Version10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison,USA), setting the following parameters: Gap weight: 8 Length weight: 2Average match: 2,912 Average mismatch: −2,003

For example a sequence which has a 80% homology with sequence SEQ ID NO:2 at the protein level is understood as meaning a sequence which, uponcomparison with the sequence SEQ ID NO: 2 by the above program algorithmwith the above parameter set, has a 80% homology.

Functional equivalents derived from one of the polypeptides as depictedin SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394 according to the invention by substitution, insertion ordeletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least55%, 60%, 65% or 70% by preference at least 80%, especially preferablyat least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably atleast 95%, 97%, 98% or 99% homology with one of the polypeptides asshown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246,248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274,276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302,304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386,388, 390, 392 or 394 according to the invention and are distinguished byessentially the same properties as the polypeptide as shown in SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308,310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or394.

Functional equivalents derived from the nucleic acid sequence asdepicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327,329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,385, 387, 389, 391 or 393 according to the invention by substitution,insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,preferably at least 55%, 60%, 65% or 70% by preference at least 80%,especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, veryespecially preferably at least 95%, 97%, 98% or 99% homology with one ofthe polypeptides as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392 or 394 according to the invention andencode polypeptides having essentially the same properties as thepolypeptide as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392 or 394.

“Essentially the same properties” of a functional equivalent is aboveall understood as meaning that the functional equivalent has abovementioned acitivty, e.g conferring an increase in the fine chemicalamount while increasing the amount of protein, activity or function ofsaid functional equivalent in an organism, e.g. a microorgansim, a plantor plant or animal tissue, plant or animal cells or a part of the same.

A nucleic acid molecule encoding an homologous to a protein sequence ofSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394 can be created by introducing one or more nucleotidesubstitutions, additions or deletions into a nucleotide sequence of thenucleic acid molecule of the present invention, in particular of SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279,281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363,365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or393 such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced into the encoding sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317,319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345,347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373,375, 377, 379, 381, 383, 385, 387, 389, 391 or 393 by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis.

Preferably, conservative amino acid substitutions are made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucinei isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Thus, a predicted nonessential amino acid residue in a polypeptide ofthe invention or a polypeptide used in the process of the invention ispreferably replaced with another amino acid residue from the samefamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a coding sequence of a nucleicacid molecule of the invention or used in the process of the invention,such as by saturation mutagenesis, and the resultant mutants can bescreened for activity described herein to identify mutants that retainor even have increased above mentioned activity, e.g. conferring anincrease in content of the fine chemical.

Following mutagenesis of one of the sequences of shown herein, theencoded protein can be expressed recombinantly and the activity of theprotein can be determined using, for example, assays described herein(see Examples).

The highest homology of the nucleic acid molecule used in the processaccording to the invention was found for the following database entriesby Gap search.

Homologues of the nucleic acid sequences used, with the sequence asdepicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327,329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,385, 387, 389, 391 or 393, comprise also allelic variants with at leastapproximately 30%, 35%, 40% or 45% homology, by preference at leastapproximately 50%, 60% or 70%, more preferably at least approximately90%, 91%, 92%, 93%, 94% or 95% and even more preferably at leastapproximately 96%, 97%, 98%, 99% or more homology with one of thenucleotide sequences shown or the abovementioned derived nucleic acidsequences or their homologues, derivatives or analogues or parts ofthese. Allelic variants encompass in particular functional variantswhich can be obtained by deletion, insertion or substitution ofnucleotides from the sequences shown, preferably from SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393, or fromthe derived nucleic acid sequences, the intention being, however, thatthe enzyme activity or the biological activity of the resulting proteinssynthesized is advantageously retained or increased.

In one embodiment of the present invention, the nucleic acid molecule ofthe invention or used in the process of the invention comprises thesequences shown in any of the sequences SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317,319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345,347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373,375, 377, 379, 381, 383, 385, 387, 389, 391 or 393. It is preferred thatthe nucleic acid molecule comprises as little as possible othernucleotides not shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179,181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291,293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319,321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,377, 379, 381, 383, 385, 387, 389, 391 or 393. In one embodiment, thenucleic acid molecule comprises less than 500, 400, 300, 200, 100, 90,80, 70, 60, 50 or 40 further nucleotides. In a further embodiment, thenucleic acid molecule comprises less than 30, 20 or 10 furthernucleotides. In one embodiment, the nucleic acid molecule use in theprocess of the invention is identical to the sequences shown in SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279,281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363,365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or393.

Also preferred is that the nucleic acid molecule used in the process ofthe invention encodes a polypeptide comprising the sequence as depictedin SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394. In one embodiment, the nucleic acid molecule encodesless than 150, 130, 100, 80, 60, 50, 40 or 30 further amino acids. In afurther embodiment, the encoded polypeptide comprises less than 20, 15,10, 9, 8, 7, 6 or 5 further amino acids. In one embodiment used in theinventive process, the encoded polypeptide is identical to the sequencesas depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326,328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,384, 386, 388, 390, 392 or 394.

In one embodiment, the nucleic acid molecule of the invention or used inthe process encodes a polypeptide comprising the sequence as depicted inSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394 and comprises less than 100 further nucleotides. In afurther embodiment, said nucleic acid molecule comprises less than 30further nucleotides. In one embodiment, the nucleic acid molecule usedin the process is identical to a coding sequence of the sequences asdepicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327,329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,385, 387, 389, 391 or 393.

Polypeptides (=proteins), which still have the essential enzymaticactivity of the polypeptide of the present invention conferring anincrease of the fine chemical i.e. whose activity is essentially notreduced, are polypeptides with at least 10% or 20%, by preference 30% or40%, especially preferably 50% or 60%, very especially preferably 80% or90 or more of the wild type biological activity or enzyme activity,advantageously, the activity is essentially not reduced in comparisonwith the activity of a polypeptide as depicted in SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370,372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 expressedunder identical conditions.

Homologues of as depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391 or 393 also mean truncated sequences,cDNA, single-stranded DNA or RNA of the coding and noncoding DNAsequence. Homologues of said sequences are also understood as meaningderivatives, which comprise noncoding regions such as, for example,UTRs, terminators, enhancers or promoter variants. The promotersupstream of the nucleotide sequences stated can be modified by one ormore nucleotide substitution(s), insertion(s) and/or deletion(s)without, however, interfering with the functionality or activity eitherof the promoters, the open reading frame (=ORF) or with the3′-regulatory region such as terminators or other 3′regulatory regions,which are far away from the ORF. It is furthermore possible that theactivity of the promoters is increased by modification of theirsequence, or that they are replaced completely by more active promoters,even promoters from heterologous organisms. Appropriate promoters areknown to the person skilled in the art and are mentioned herein below.

In a further embodiment, the process according to the present inventioncomprises the following steps:

-   (a) selecting an organism or a part thereof expressing the    polypeptide of this invention;-   (b) mutagenizing the selected organism or the part thereof;-   (c) comparing the activity or the expression level of said    polypeptide in the mutagenized organism or the part thereof with the    activity or the expression of said polypeptide in the selected    organisms or the part thereof;-   (d) selecting the mutagenized organisms or parts thereof, which    comprise an increased activity or expression level of said    polypeptide compared to the selected organism (a) or the part    thereof;-   (e) optionally, growing and cultivating the organisms or the parts    thereof; and-   (f) recovering, and optionally isolating, the free or bound the fine    chemical produced by the selected mutated organisms or parts    thereof.

The organisms or part thereof produce according to the herein mentionedprocess of the invention an increased level of free and/or protein-boundfine chemical compared to said control or selected organisms or partsthereof.

Advantageously the seclected organisms are mutagenized according to theinvention. According to the invention mutagenesis is any change of thegenetic information in the genom of an organism, that means anystructural or compositional change in the nucleic acid preferably DNA ofan organism that is not caused by normal segregation or geneticrecombiantion processes. Such mutations may occur spontaneously, or maybe induced by mutagens as described below. Such change can be inducedeither randomly or selectivly. In both cases the genetic information ofthe organism is modified. In general this lead to the situation that theactivity of the gene product of the relevant genes inside the cells orinside the organism is increased.

In case of the specific or so called site directed mutagenesis adistinct gene is mutated and thereby its activity and/or the activity orthe encoded gene product is repressed, reduced or increased, preferablyincreased. In the event of a random mutagenesis one or more genes aremutated by chance and their activities and/or the activities of theirgene productes are repressed, reduced or increased, preferablyincreased.

For the purpose of a mutagenesis of a huge population of organisms, suchpopulation can be transformed with a DNA construct, which is useful forthe activation of as much as possible genes of an organism, preferablyall genes. For example the construct can contain a strong promoter orone or more enhancers, which are capable of transcriptionally activategenes in the vicinity of their integration side. With this method it ispossible to statistically mutagenize, eg activate nearly all genes of anorganism by the random integration of an activation construct.Afterwards the skilled worker can identify those mutagenized lines inwhich a gene of the invention has been activated, which in turns leadsto the desired increase in the fine chemical production.

The genes of the invention can also be activated by mutagensis, eitherof regulatory or coding regions. In the event of a random mutagenesis ahuge number of organisms are treated with a mutagenic agent. The amountof said agent and the intensity of the treatment will be chosen in sucha manner that statistically nearly every gene is mutated once. Theprocess for the random mutagensis as well as the respective agens iswell known by the skilled person. Such methods are disclosed for exampleby A. M. van Harten [(1998), “Mutation breeding: theory and practicalapplications”, Cambridge University Press, Cambridge, UK], E Friedberg,G Walker, W Siede [(1995), “DNA Repair and Mutagenesis”, BlackwellPublishing], or K. Sankaranarayanan, J. M. Gentile, L. R. Ferguson[(2000), “Protocols in Mutagenesis”, Elsevier Health Sciences]. As theskilled worker knows the spontaneous mutation rate in the cells of anorganism is very low and that a large numer of chemical, physical orbiological agents are available for the mutagenesis of organisms. Theseagents are named as mutagens or mutagenic agents. As mentioned beforethree different kinds of mutagens (chemical, physical or biologicalagents) are available.

There are different classes of chemical mutagens, which can be separatedby their mode of action. For example base analogues such as5-bromouracil, 2-amino purin. Other chemical mutagens are interactingwith the DNA such as sulphuric acid, nitrous acid, hydroxylamine; orother alkylating agents such as monofunctional agents like ethylmethanesulfonate, dimethylsulfate, methyl methanesulfonate),bifunctional like dichloroethyl sulphide, Mitomycin,Nitrosoguanidine-dialkylnitrosamine, N-Nitrosoguanidin derivatives,N-alkyl-N-nitro-N-nitroso-guanidine-), ntercalating dyes like Acridine,ethidium bromide).

Physical mutagens are for example ionizing irradiation (X ray), UVirradiation. Different forms of irradiation are available and they arestrong mutagens. Two main classes of irradiation can be distinguished:a) non-ionizing irradiation such as UV light or ionizing irradiationsuch as X ray. Biological mutagens are for example transposable elementsfor example IS elements such as IS100, transposons such as Tn5, Tn10,Tn916 or Tn1000 or phages like Mu^(amplac), P1, T5, λplac etc. Methodsfor introducing this phage DNA into the appropriate microorganism arewell known to the skilled worker (see Microbiology, Third Edition, Eds.Davis, B. D., Dulbecco, R., Eisen, H. N. and Ginsberg, H. S., HarperInternational Edition, 1980). The common procedure of a transposonmutagenesis is the insertion of a transposable element within a gene ornearby for example in the promotor or terminator region and therebyleading to a loss of the gene function. Procedures to localize thetransposon within the genome of the organisms are well known by a personskilled in the art.

Preferably a chemical or biochemical procedure is used for themutagenesis of the organisms. A preferred chemical method is themutagensis with N-methyl-N-nitro-nitrosoguanidine.

Other biological methods are disclosed by Spee et al. (Nucleic AcidsResearch, Vol. 21, No. 3, 1993: 777-778). Spee et al. teaches a PCRmethod using dITP for the random mutagenesis. This method described bySpee et al. was further improved by Rellos et al. (Protein Expr. Purif.,5, 1994: 270-277). The use of an in vitro recombination technique formolecular mutagenesis is described by Stemmer (Proc. Natl. Acad. Sci.USA, Vol. 91, 1994: 10747-10751). Moore et al. (Nature BiotechnologyVol. 14, 1996: 458-467) describe the combination of the PCR andrecombination methods for increasing the enzymatic activity of anesterase toward a para-nitrobenzyl ester. Another route to themutagenesis of enzymes is described by Greener et al. in Methods inMolecular Biology (Vol. 57, 1996: 375-385). Greener et al. use thespecific Escherichia coli strain XL1-Red to generate Escherichia colimutants, which have increased antibiotic resistance.

In one embodiment, the protein according to the invention or the nucleicacid molecule characterized herein originates from a eukaryotic orprokaryotic organism such as a non-human animal, a plant, amicroorganism such as a fungus, yeast, an alga, a diatom or a bacterium.Nucleic acid molecules, which advantageously can be used in the processof the invention originate from yeasts, for example the familySaccharomycetaceae, in particular the genus Saccharomyces, or yeastgenera such as Candida, Hansenula, Pichia, Yarrowia, Rhodotorula orSchizosaccharomyces and the especially advantageous from the speciesSaccharomyces cerevisiae.

In one embodiment, nucleic acid molecules, which advantageously can beused in the process of the invention orginate from yeast, for examplefrom Saccharomycetaceae, particularly from the genus Saccharomycesadvantageously form the species Saccharomyces cerevisiae.

If, in the process according to the invention, plants are selected asthe donor organism, this plant may, in principle, be in any phylogeneticrelation of the recipient plant. Donor and recipient plant may belong tothe same family, genus, species, variety or line, resulting in anincreasing homology between the nucleic acids to be integrated andcorresponding parts of the genome of the recipient plant. This alsoapplies analogously to microorganisms as donor and recipient organism.It might also be advantageously to use nuclei acids molecules from verydistinct species, since these might exhibit reduced sensitivity againstendogenous regulatory mechanisms and such sequences might not berecognized by endogenous silencing mechanisms.

Accordingly, one embodiment of the application relates to the use ofnucleic acid molecules in the process of the invention from plants, e.g.crop plants, e.g. from: B. napus; O. sativa, Glycine max; B. vulgaris,L. japonicus, Z. elegans, Z. mays, C. arietinum, A. thaliana, H.vulgare, N. tabacum, G. hirsutum, P. patens, F. distichus, sunflowerlinseed or maize or their homologues.

Accordingly, in one embodiment, the invention relates to a nucleic acidmolecule, which comprises a nucleic acid molecule selected from thegroup consisting of:

-   a) nucleic acid molecule encoding of the polypeptide as depicted in    SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,    32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72,    74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,    106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,    132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,    158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,    184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,    210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,    236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,    262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,    288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,    314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,    340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,    366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392    or 394 or a fragment thereof, which confers an increase in the    amount of fine chemical in an organism or a part thereof;-   b) nucleic acid molecule comprising of the nucleic acid molecule as    depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,    25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65,    67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,    101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,    127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,    153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,    179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,    205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,    231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,    257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281,    283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,    309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,    335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359,    361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385,    387, 389, 391 or 393 or a fragment thereof, which confers an    increase in the amount of fine chemical in an organism or a part    thereof;-   c) nucleic acid molecule whose sequence can be deduced from a    polypeptide sequence encoded by a nucleic acid molecule of (a)    or (b) as a result of the degeneracy of the genetic code and    conferring an increase in the amount of fine chemical in an organism    or a part thereof;-   d) nucleic acid molecule which encodes a polypeptide which has at    least 50% identity with the amino acid sequence of the polypeptide    encoded by the nucleic acid molecule of (a) to (c) and conferring an    increase in the amount of fine chemical in an organism or a part    thereof;-   e) nucleic acid molecule which hybridizes with a nucleic acid    molecule of (a) to (c) under stringent hybridization conditions and    conferring an increase in the amount of fine chemical in an organism    or a part thereof;-   f) nucleic acid molecule encoding a polypeptide, the polypeptide    being derived by substituting, deleting and/or adding one or more    amino acids of the amino acid sequence of the polypeptide encoded by    the nucleic acid molecules (a) to (d), preferably to (a) to (c), and    conferring an increase in the amount of the fine chemical in an    organism or a part thereof;-   g) nucleic acid molecule encoding a fragment or an epitope of a    polypeptide which is encoded by one of the nucleic acid molecules    of (a) to (e), preferably to (a) to (c) and conferring an increase    in the amount of the fine chemical in an organism or a part thereof;-   h) nucleic acid molecule which encompasses a nucleic acid molecule    which is obtained by amplifying nucleic acid molecules from a cDNA    library or a genomic library using the primers in SEQ ID NO: 53, SEQ    ID NO: 54, SEQ ID NO: 395 or SEQ ID NO: 396 and conferring an    increase in the amount of the fine chemical in an organism or a part    thereof;-   i) nucleic acid molecule encoding a polypeptide which is isolated,    e.g. from a expression library, with the aid of monoclonal and/or    polyclonal antibodies against a polypeptide encoded by one of the    nucleic acid molecules of (a) to (g), preferably to (a) to (c) and    conferring an increase in the amount of the fine chemical in an    organism or a part thereof;-   j) nucleic acid molecule encoding a polypeptide comprising the    consensus sequence as depicted in SEQ ID NO: 47, SEQ ID NO: 48, SEQ    ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:    397, SEQ ID NO: 398, SEQ ID NO: 399 and/or SEQ ID NO: 400 and    conferring an increase in the amount of the fine chemical in an    organism or a part thereof; and/or-   k) nucleic acid molecule which is obtainable by screening a suitable    nucleic acid library under stringent hybridization conditions with a    probe comprising one of the sequences of the nucleic acid molecule    of (a) to (k) or with a fragment of at least 15 nt, preferably 20    nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid    molecule characterized in (a) to (h) or of the nucleic acid molecule    as depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,    25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65,    67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,    101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,    127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,    153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,    179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,    205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,    231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,    257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281,    283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,    309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,    335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359,    361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385,    387, 389, 391 or 393 or a nucleic acid molecule encoding, preferably    at least the mature form of, the polypeptide as depicted in SEQ ID    NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,    36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,    78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,    110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,    136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,    162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,    188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,    214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,    240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,    266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,    292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,    318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,    344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,    370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394,    and conferring an increase in the amount of the fine chemical in an    organism or a part thereof;    or which encompasses a sequence which is complementary thereto;    whereby, preferably, the nucleic acid molecule according to (a)    to (k) distinguishes over the sequence as depicted in SEQ ID NO: 1,    3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,    39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,    81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,    111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,    137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,    163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,    189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,    215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,    241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,    267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291,    293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317,    319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343,    345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,    371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393 by one    or more nucleotides. In one embodiment, the nucleic acid molecule of    the invention does not consist of the sequence as depicted in SEQ ID    NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,    35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,    77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,    109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,    135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,    161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,    187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,    213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,    239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,    265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,    291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315,    317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,    343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367,    369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393.    In an other embodiment, the nucleic acid molecule of the present    invention is at least 30% identical and less than 100%, 99.999%,    99.99%, 99.9% or 99% identical to the sequence as depicted in SEQ ID    NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,    35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,    77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,    109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,    135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,    161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,    187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,    213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,    239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263,    265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,    291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315,    317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,    343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367,    369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393.    In a further embodiment the nucleic acid molecule does not encode    the polypeptide sequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10,    12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,    46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,    88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,    118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,    144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,    170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,    196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,    222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246,    248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,    274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,    300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,    326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,    352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,    378, 380, 382, 384, 386, 388, 390, 392 or 394. Accordingly, in one    embodiment, the nucleic acid molecule of the present invention    encodes in one embodiment a polypeptide which differs at least in    one or more amino acids from the polypeptide as depicted in SEQ ID    NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,    36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,    78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,    110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,    136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,    162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,    188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,    214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,    240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,    266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,    292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,    318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,    344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,    370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394.    In another embodiment, the nucleic acid molecule as depicted in SEQ    ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,    33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,    75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,    107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,    133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,    159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,    185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,    211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,    237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,    263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287,    289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,    315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339,    341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365,    367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or    393 does not encode a protein of the sequence as depicted in SEQ ID    NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,    36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,    78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,    110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,    136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,    162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,    188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,    214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,    240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,    266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,    292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,    318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,    344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,    370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394.    Accordingly, in one embodiment, the protein encoded by a sequences    of a nucleic acid accoriding to (a) to (k) does not consist of the    sequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,    20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60,    62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,    96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,    124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,    150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,    176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,    202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,    228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,    254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,    280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,    306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,    332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,    358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,    384, 386, 388, 390, 392 or 394. In a further embodiment, the protein    of the present invention is at least 30% identical to protein    sequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,    20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60,    62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,    96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,    124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,    150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,    176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,    202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,    228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,    254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,    280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,    306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,    332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,    358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,    384, 386, 388, 390, 392 or 394 and less than 100%, preferably less    than 99.999%, 99.99% or 99.9%, more preferably less than 99%, 98%,    97%, 96% or 95% identical to the sequence as depicted in SEQ ID NO:    2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,    38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,    80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,    110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,    136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,    162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,    188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,    214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,    240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,    266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,    292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,    318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,    344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,    370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394.

The nucleic acid sequences used in the process are advantageouslyintroduced in a nucleic acid construct, preferably an expressioncassette, which makes the expression of the nucleic acid molecules in anorganism, advantageously a plant or a microorganism possible.

Accordingly, the invention also relates to a nucleic acid construct,preferably to an expression construct, comprising the nucleic acidmolecule of the present invention functionally linked to one or moreregulatory elements or signals.

As described herein, the nucleic acid construct can also comprisefurther genes, which are introduced into the organisms or cells. It ispossible and advantageous to introduce into, and express in, the hostorganisms regulatory genes such as genes for inductors, repressors orenzymes, which, owing to their enzymatic activity, engage in theregulation of one or more genes of a biosynthetic pathway. These genescan be of heterologous or homologous origin. Moreover, furtherbiosynthesis genes may advantageously be present, or else these genesmay be located on one or more further nucleic acid constructs. Genes,which are advantageously employed as biosynthesis genes are genes of theamino acid metabolism, of glycolysis, of the tricarboxylic acidmetabolism or their combinations. As described herein, regulatorsequences or factors can have a positive effect on preferably the geneexpression of the genes introduced, thus increasing it. Thus, anenhancement of the regulator elements may advantageously take place atthe transcriptional level by using strong transcription signals such aspromoters and/or enhancers. In addition, however, an enhancement oftranslation is also possible, for example by increasing mRNA stabilityor by inserting a translation enhancer sequence.

In principle, the nucleic acid construct can comprise the hereindescribed regulator sequences and further sequences relevant for theexpression of the comprised genes. Thus, the nucleic acid construct ofthe invention can be used as expression cassette and thus can be useddirectly for introduction into the plant, or else they may be introducedinto a vector. Accordingly in one embodiment the nucleic acid constructis an expression cassette comprising a microorganism promoter or amicroorganism terminator or both. In another embodiment the expressioncassette encompasses a plant promoter or a plant terminator or both.

Accordingly, in one embodiment, the process according to the inventioncomprises the following steps:

-   (a) introducing of a nucleic acid construct comprising the nucleic    acid molecule of the invention or used in the process of the    invention or encoding the polypeptide of the present invention or    used in the process of the invention; or-   (b) introducing of a nucleic acid molecule, including regulatory    sequences or factors, which expression increases the expression of    the nucleic acid molecule of the invention or used in the process of    the invention or encoding the polypeptide of the present invention    or used in the process of the invention in a cell, or an organism or    a part thereof, preferably in a plant, plant cell or a    microorganism, and-   (c) expressing of the gene product encoded by the nucleic acid    construct or the nucleic acid molecule mentioned under (a) or (b) in    the cell or the organism.

After the introduction and expression of the nucleic acid construct thetransgenic organism or cell is advantageously cultured and subsequentlyharvested. The transgenic organism or cell may be a prokaryotic oreukaryotic organism such as a microorganism, a non-human animal andplant for example a plant or animal cell, a plant or animal tissue,preferably a crop plant, or a part thereof.

To introduce a nucleic acid molecule into a nucleic acid construct, e.g.as part of an expression cassette, the codogenic gene segment isadvantageously subjected to an amplification and ligation reaction inthe manner known by a skilled person. It is preferred to follow aprocedure similar to the protocol for the Pfu DNA polymerase or aPfu/Taq DNA polymerase mixture. The primers are selected according tothe sequence to be amplified. The primers should expediently be chosenin such a way that the amplificate comprise the codogenic sequence fromthe start to the stop codon. After the amplification, the amplificate isexpediently analyzed. For example, the analysis may consider quality andquantity and be carried out following separation by gel electrophoresis.Thereafter, the amplificate can be purified following a standardprotocol (for example Qiagen). An aliquot of the purified amplificate isthen available for the subsequent cloning step. The skilled workergenerally knows suitable cloning vectors.

They include, in particular, vectors which are capable of replication ineasy to handle cloning systems like as bacterial yeast or insect cellbased (e.g. baculovirus expression) systems, that is to say especiallyvectors which ensure efficient cloning in E. coli, and which makepossible the stable transformation of plants. Vectors, which must bementioned, in particular are various binary and cointegrated vectorsystems, which are suitable for the T-DNA-mediated transformation. Suchvector systems are generally characterized in that they contain at leastthe vir genes, which are required for the Agrobacterium-mediatedtransformation, and the T-DNA border sequences.

In general, vector systems preferably also comprise furthercis-regulatory regions such as promoters and terminators and/orselection markers by means of which suitably transformed organisms canbe identified. While vir genes and T-DNA sequences are located on thesame vector in the case of cointegrated vector systems, binary systemsare based on at least two vectors, one of which bears vir genes, but noT-DNA, while a second one bears T-DNA, but no vir gene. Owing to thisfact, the last-mentioned vectors are relatively small, easy tomanipulate and capable of replication in E. coli and in Agrobacterium.These binary vectors include vectors from the series pBIB-HYG, pPZP,pBecks, pGreen. Those, which are preferably used in accordance with theinvention, are Bin19, pBI101, pBinAR, pGPTV and pCAMBIA. An overview ofbinary vectors and their use is given by Hellens et al, Trends in PlantScience (2000) 5, 446-451.

For a vector preparation, vectors may first be linearized usingrestriction endonuclease(s) and then be modified enzymatically in asuitable manner. Thereafter, the vector is purified, and an aliquot isemployed in the cloning step. In the cloning step, the enzyme-cleavedand, if required, purified amplificate is cloned together with similarlyprepared vector fragments, using ligase. In this context, a specificnucleic acid construct, or vector or plasmid construct, may have one orelse more codogenic gene segments. The codogenic gene segments in theseconstructs are preferably linked operably to regulatory sequences. Theregulatory sequences include, in particular, plant sequences like theabove-described promoters and terminators. The constructs canadvantageously be propagated stably in microorganisms, in particularEscherichia coli and/or Agrobacterium tumefaciens, under selectiveconditions and enable the transfer of heterologous DNA into plants orother microorganisms. In accordance with a particular embodiment, theconstructs are based on binary vectors (overview of a binary vector:Hellens et al., 2000). As a rule, they contain prokaryotic regulatorysequences, such as replication origin and selection markers, for themultiplication in microorganisms such as Escherichia coli andAgrobacterium tumefaciens. Vectors can further contain agrobacterialT-DNA sequences for the transfer of DNA into plant genomes or othereukaryotic regulatory sequences for transfer into other eukaryoticcells, e.g. Saccharomyces sp. or other prokaryotic regulatory sequencesfor the transfer into other prokaryotic cells, e.g. Corynebacterium sp.or Bacillus sp. For the transformation of plants, the right bordersequence, which comprises approximately 25 base pairs, of the totalagrobacterial T-DNA sequence is advantageously included. Usually, theplant transformation vector constructs according to the inventioncontain T-DNA sequences both from the right and from the left borderregion, which contain expedient recognition sites for site-specificacting enzymes, which, in turn, are encoded by some of the vir genes.

Suitable host organisms are known to the skilled worker. Advantageousorganisms are described further above in the present application. Theyinclude in particular eukaryotes or eubacteria, e.g. prokaryotes orarchae bacteria. Advantageously host organisms are microorganismsselected from the group consisting of Actinomycetaceae, Bacillaceae,Brevibacteriaceae, Corynebacteriaceae, Enterobacteriacae, Gordoniaceae,Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pseudomonaceae,Rhizobiaceae, Streptomycetaceae, Chaetomiaceae, Choanephoraceae,Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae,Mortierellaceae, Mucoraceae, Pythiaceae, Saccharomycetaceae,Saprolegniaceae, Schizosaccharomycetaceae, Sodariaceae,Sporobolomycetaceae, Tuberculariaceae, Adelotheciaceae, Dinophyceae,Ditrichaceae and Prasinophyceae. Preferably are unicellular,microorganisms, e.g. fungi, bacteria or protoza, such as fungi like thegenus Claviceps or Aspergillus or gram-positive bacteria such as thegenera Bacillus, Corynebacterium, Micrococcus, Brevibacterium,Rhodococcus, Nocardia, Caseobacter or Arthrobacter or gram-negativebacteria such as the genera Escherichia, Flavobacterium or Salmonella,or yeasts such as the genera Rhodotorula, Hansenula, Pichia, Yerrowia,Saccharomyces, Schizosaccharomyces or Candida.

Host organisms which are especially advantageously selected in theprocess according to the invention are microorganisms selected from thegroup of the genera and species consisting of Hansenula anomala,Saccharomyces cerevisiae, Candida utilis, Claviceps purpurea, Bacilluscirculans, Bacillus subtilis, Bacillus sp., Brevibacterium albidum,Brevibacterium album, Brevibacterium cerinum, Brevibacterium flavum,Brevibacterium glutamigenes, Brevibacterium iodinum, Brevibacteriumketoglutamicum, Brevibacterium lactofermentum, Brevibacterium linens,Brevibacterium roseum, Brevibacterium saccharolyticum, Brevibacteriumsp., Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum,Corynebacterium ammoniagenes, Corynebacterium glutamicum (=Micrococcusglutamicum), Corynebacterium melassecola, Corynebacterium sp. orEscherichia coli, specifically Saccharomyces cerevisiae or Escherichiacoli K12 and its described strains.

Advantageously preferred in accordance with the invention are hostorganisms of the genus Agrobacterium tumefaciens or plants. Preferredplants are selected from among the families Aceraceae, Anacardiaceae,Apiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae, Brassicaceae,Bromeliaceae, Cactaceae, Caricaceae, Caryophyllaceae, Cannabaceae,Convolvulaceae, Chenopodiaceae, Elaeagnaceae, Geraniaceae, Gramineae,Juglandaceae, Lauraceae, Leguminosae, Linaceae, Cucurbitaceae,Cyperaceae, Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae,Papaveraceae, Rosaceae, Salicaceae, Solanaceae, Arecaceae, Iridaceae,Liliaceae, Orchidaceae, Gentianaceae, Labiaceae, Magnoliaceae,Ranunculaceae, Carifolaceae, Rubiaceae, Scrophulariaceae, Ericaceae,Polygonaceae, Violaceae, Juncaceae, Poaceae, perennial grass, foddercrops, vegetables and ornamentals.

Especially preferred are plants selected from the groups of the familiesApiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae,Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae. Especiallyadvantageous are, in particular, crop plants. Accordingly, anadvantageous plant preferably belongs to the group of the genus peanut,oilseed rape, canola, sunflower, safflower, olive, sesame, hazelnut,almond, avocado, bay, pumpkin/squash, linseed, soya, pistachio, borage,maize, wheat, rye, oats, sorghum and millet, triticale, rice, barley,cassava, potato, sugarbeet, fodder beet, egg plant, and perennialgrasses and forage plants, oil palm, vegetables (brassicas, rootvegetables, tuber vegetables, pod vegetables, fruiting vegetables, onionvegetables, leafy vegetables and stem vegetables), buckwheat, Jerusalemartichoke, broad bean, vetches, lentil, alfalfa, dwarf bean, lupin,clover and lucerne.

In order to introduce, into a plant, the nucleic acid molecule of theinvention or used in the process according to the invention, it hasproved advantageous first to transfer them into an intermediate host,for example a bacterium or a eukaryotic unicellular cell. Thetransformation into E. coli, which can be carried out in a manner knownper se, for example by means of heat shock or electroporation, hasproved itself expedient in this context. Thus, the transformed E. colicolonies can be analysed for their cloning efficiency. This can becarried out with the aid of a PCR. Here, not only the identity, but alsothe integrity, of the plasmid construct can be verified with the aid ofa defined colony number by subjecting an aliquot of the colonies to saidPCR. As a rule, universal primers which are derived from vectorsequences are used for this purpose, it being possible, for example, fora forward primer to be arranged upstream of the start ATG and a reverseprimer to be arranged downstream of the stop codon of the codogenic genesegment. The amplificates are separated by electrophoresis and assessedwith regard to quantity and quality.

The nucleic acid constructs, which are optionally verified, aresubsequently used for the transformation of the plants or other hosts,e.g. other eukaryotic cells or other prokaryotic cells. To this end, itmay first be necessary to obtain the constructs from the intermediatehost. For example, the constructs may be obtained as plasmids frombacterial hosts by a method similar to conventional plasmid isolation.

The nucleic acid molecule of the invention or used in the processaccording to the invention can also be introduced into modified viralvectors like baculovirus vectors for expression in insect cells or plantviral vectors like tobacco mosaic virus or potato virus X-based vectors.Approaches leading to the expression of proteins from the modified viralgenome including the the nucleic acid molecule of the invention or usedin the process according to the invention involve for example theinoculation of tobacco plants with infectious RNA transcribed in vitrofrom a cDNA copy of the recombinant viral genome. Another approachutilizes the transfection of whole plants from wounds inoculated withAgrobacterium tumefaciens containing cDNA copies of recombinantplus-sense RNA viruses. Different vectors and virus are known to theskilled worker for expression in different target eg. production plants.

A large number of methods for the transformation of plants are known.Since, in accordance with the invention, a stable integration ofheterologous DNA into the genome of plants is advantageous, theT-DNA-mediated transformation has proved expedient in particular. Forthis purpose, it is first necessary to transform suitable vehicles, inparticular agrobacteria, with a codogenic gene segment or thecorresponding plasmid construct comprising the nucleic acid molecule ofthe invention. This can be carried out in a manner known per se. Forexample, said nucleic acid construct of the invention, or saidexpression construct or said plasmid construct, which has been generatedin accordance with what has been detailed above, can be transformed intocompetent agrobacteria by means of electroporation or heat shock. Inprinciple, one must differentiate between the formation of cointegratedvectors on the one hand and the transformation with binary vectors onthe other hand. In the case of the first alternative, the constructs,which comprise the codogenic gene segment or the nucleic acid moleculeof the invention have no T-DNA sequences, but the formation of thecointegrated vectors or constructs takes place in the agrobacteria byhomologous recombination of the construct with T-DNA. The T-DNA ispresent in the agrobacteria in the form of Ti or Ri plasmids in whichexogenous DNA has expediently replaced the oncogenes. If binary vectorsare used, they can be transferred to agrobacteria either by bacterialconjugation or by direct transfer. These agrobacteria expedientlyalready comprise the vector bearing the vir genes (currently referred toas helper Ti(Ri) plasmid).

One or more markers may expediently also be used together with thenucleic acid construct, or the vector of the invention and, if plants orplant cells shall be transformed together with the T-DNA, with the aidof which the isolation or selection of transformed organisms, such asagrobacteria or transformed plant cells, is possible. These marker genesenable the identification of a successful transfer of the nucleic acidmolecules according to the invention via a series of differentprinciples, for example via visual identification with the aid offluorescence, luminescence or in the wavelength range of light which isdiscernible for the human eye, by a resistance to herbicides orantibiotics, via what are known as nutritive markers (auxotrophismmarkers) or antinutritive markers, via enzyme assays or viaphytohormones. Examples of such markers which may be mentioned are GFP(=green fluorescent protein); the luciferin/luceferase system, theβ-galactosidase with its colored substrates, for example X-Gal, theherbicide resistances to, for example, imidazolinone, glyphosate,phosphinothricin or sulfonylurea, the antibiotic resistances to, forexample, bleomycin, hygromycin, streptomycin, kanamycin, tetracyclin,chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycinor blasticidin, to mention only a few, nutritive markers such as theutilization of mannose or xylose, or antinutritive markers such as theresistance to 2-deoxyglucose. This list is a small number of possiblemarkers. The skilled worker is very familiar with such markers.Different markers are preferred, depending on the organism and theselection method.

As a rule, it is desired that the plant nucleic acid constructs areflanked by T-DNA at one or both sides of the codogenic gene segment.This is particularly useful when bacteria of the species Agrobacteriumtumefaciens or Agrobacterium rhizogenes are used for the transformation.A method, which is preferred in accordance with the invention, is thetransformation with the aid of Agrobacterium tumefaciens. However,biolistic methods may also be used advantageously for introducing thesequences in the process according to the invention, and theintroduction by means of PEG is also possible. The transformedagrobacteria can be grown in the manner known per se and are thusavailable for the expedient transformation of the plants. The plants orplant parts to be transformed are grown or provided in the customarymanner. The transformed agrobacteria are subsequently allowed to act onthe plants or plant parts until a sufficient transformation rate isreached. Allowing the agrobacteria to act on the plants or plant partscan take different forms. For example, a culture of morphogenic plantcells or tissue may be used. After the T-DNA transfer, antibiotics as arule eliminate the bacteria, and the regeneration of plant tissue isinduced. This is done in particular using suitable plant hormones inorder to initially induce callus formation and then to promote shootdevelopment.

The transfer of foreign genes into the genome of a plant is calledtransformation. In doing this the methods described for thetransformation and regeneration of plants from plant tissues or plantcells are utilized for transient or stable transformation. Anadvantageous transformation method is the transformation in planta. Tothis end, it is possible, for example, to allow the agrobacteria to acton plant seeds or to inoculate the plant meristem with agrobacteria. Ithas proved particularly expedient in accordance with the invention toallow a suspension of transformed agrobacteria to act on the intactplant or at least the flower primordia. The plant is subsequently grownon until the seeds of the treated plant are obtained (Clough and Bent,Plant J. (1998) 16, 735-743). To select transformed plants, the plantmaterial obtained in the transformation is, as a rule, subjected toselective conditions so that transformed plants can be distinguishedfrom untransformed plants. For example, the seeds obtained in theabove-described manner can be planted and, after an initial growingperiod, subjected to a suitable selection by spraying. A furtherpossibility consists in growing the seeds, if appropriate aftersterilization, on agar plates using a suitable selection agent so thatonly the transformed seeds can grow into plants. Further advantageoustransformation methods, in particular for plants, are known to theskilled worker and are described hereinbelow.

Further advantageous and suitable methods are protoplast transformationby poly (ethylene glycol)-induced DNA uptake, the “biolistic” methodusing the gene cannon—referred to as the particle bombardment method,electroporation, the incubation of dry embryos in DNA solution,microinjection and gene transfer mediated by Agrobacterium. Said methodsare described by way of example in B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol.1, Engineering and Utilization,eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in PotrykusAnnu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). Thenucleic acids or the construct to be expressed is preferably cloned intoa vector, which is suitable for transforming Agrobacterium tumefaciens,for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).Agrobacteria transformed by such a vector can then be used in knownmanner for the transformation of plants, in particular of crop plantssuch as by way of example tobacco plants, for example by bathing bruisedleaves or chopped leaves in an agrobacterial solution and then culturingthem in suitable media. The transformation of plants by means ofAgrobacterium tumefaciens is described, for example, by Höfgen andWillmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter aliafrom F. F. White, Vectors for Gene Transfer in Higher Plants; inTransgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kungand R. Wu, Academic Press, 1993, pp. 15-38.

The abovementioned nucleic acid molecules can be cloned into the nucleicacid constructs or vectors according to the invention in combinationtogether with further genes, or else different genes are introduced bytransforming several nucleic acid constructs or vectors (includingplasmids) into a host cell, advantageously into a plant cell or amicroorgansims.

In addition to the sequence mentioned in SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317,319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345,347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373,375, 377, 379, 381, 383, 385, 387, 389, 391 or 393 or its derivatives,it is advantageous additionally to express and/or mutate further genesin the organisms. Especially advantageously, additionally at least onefurther gene of the fine chemical biosynthetic pathway e.g. the aminoacid biosynthetic pathway such as for L-tryptophane, L-isoleucine,L-leucine, L-lysine, L-threonine and/or L-methionine to mention only acouple of them is expressed in the organisms such as plants ormicroorganisms. It is also possible that the regulation of the naturalgenes has been modified advantageously so that the gene and/or its geneproduct is no longer subject to the regulatory mechanisms which exist inthe organisms. This leads to an increased synthesis of the finechemicals e.g. the amino acids desired since, for example, feedbackregulations no longer exist to the same extent or not at all. Inaddition it might be advantageously to combine the sequences shown inSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249,251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361,363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389,391 or 393 with genes which generally support or enhances to growth oryield of the target organismen, for example genes which lead to fastergrowth rate of microorganisms or genes which produces stress-, pathogen,or herbicide resistant plants.

In a further embodiment of the process of the invention, therefore,organisms are grown, in which there is simultaneous overexpression of atleast one nucleic acid or one of the genes which code for proteinsinvolved in the fine chemical metabolism e.g. the amino acid metabolism,in particular in amino acid synthesis.

A further advantageous nucleic acid sequence which can be expressed incombination with the sequences used in the process and/or theabovementioned biosynthesis genes is the sequence of the ATP/ADPtranslocator as described in WO 01/20009. This ATP/ADP translocatorleads to an increased synthesis of the essential amino acids lysineand/or methionine. Furthermore, an advantageous nucleic acid sequencecoexpressed can be threonine adlolase and/or lysine decarboxylase asdescribed in the state of the art.

In a further advantageous embodiment of the process of the invention,the organisms used in the process are those in which simultaneously atleast one of the aforementioned genes or one of the aforementionednucleic acids is mutated so that the activity of the correspondingproteins is influenced by metabolites to a smaller extent compared withthe unmutated proteins, or not at all, and that in particular theproduction according to the invention of the fine chemical for exampleof the amino acids is not impaired, or so that their specific enzymaticactivity is increased. Less influence means in this connection that theregulation of the enzymic activity is less by at least 10%,advantageously at least 20, 30 or 40%, particularly advantageously by atleast 50, 60 or 70%, compared with the starting organism, and thus theactivity of the enzyme is increased by these figures mentioned comparedwith the starting organism. An increase in the enzymatic activity meansan enzymatic activity which is increased by at least 10%, advantageouslyat least 20, 30 or 40%, particularly advantageously by at least 50, 60or 70%, compared with the starting organism. This leads to an increasedproductivity of the fine chemical.

In a further advantageous embodiment of the process of the invention,the organisms used in the process are those in which simultaneously thefine chemical degrading protein is attenuated, in particular by reducingthe rate of expression of the corresponding gene.

In another embodiment of the process of the invention, the organismsused in the process are those in which simultaneously at least one ofthe aforementioned nucleic acids or of the aforementioned genes ismutated in such a way that the enzymatic activity of the correspondingprotein is partially reduced or completely blocked. A reduction in theenzymatic activity means an enzymatic activity, which is reduced by atleast 10%, advantageously at least 20, 30 or 40%, particularlyadvantageously by at least 50, 60 or 70%, preferably more, compared withthe starting organism.

If it is intended to transform the host cell, in particular the plantcell, with several constructs or vectors, the marker of a precedingtransformation must be removed or a further marker employed in afollowing transformation. The markers can be removed from the host cell,in particular the plant cell, as described hereinbelow via methods withwhich the skilled worker is familiar. In particular plants without amarker, in particular without resistance to antibiotics, are anespecially preferred embodiment of the present invention.

In the process according to the invention, the nucleic acid sequencesused in the process according to the invention are advantageously linkedoperably to one or more regulatory signals in order to increase geneexpression. These regulatory sequences are intended to enable thespecific expression of the genes and the expression of protein.Depending on the host organism for example plant or microorganism, thismay mean, for example, that the gene is expressed and/or overexpressedafter induction only, or that it is expressed and/or overexpressedconstitutively. These regulatory sequences are, for example, sequencesto which the inductors or repressors bind and which thus regulate theexpression of the nucleic acid. In addition to these novel regulatorysequences, or instead of these sequences, the natural regulation ofthese sequences may still be present before the actual structural genesand, if appropriate, may have been genetically modified so that thenatural regulation has been switched off and gene expression has beenincreased. However, the nucleic acid construct of the invention suitableas expression cassette (=expression construct=gene construct) can alsobe simpler in construction, that is to say no additional regulatorysignals have been inserted before the nucleic acid sequence or itsderivatives, and the natural promoter together with its regulation hasnot been removed. Instead, the natural regulatory sequence has beenmutated in such a way that regulation no longer takes place and/or geneexpression is increased. These modified promoters can also be introducedon their own before the natural gene in the form of part sequences(=promoter with parts of the nucleic acid sequences according to theinvention) in order to increase the activity. Moreover, the geneconstruct can advantageously also comprise one or more of what are knownas enhancer sequences in operable linkage with the promoter, and theseenable an increased expression of the nucleic acid sequence. Also, it ispossible to insert additional advantageous sequences at the 3′ end ofthe DNA sequences, such as, for example, further regulatory elements orterminators.

The nucleic acid molecules, which encode proteins according to theinvention and nucleic acid molecules, which encode other polypeptidesmay be present in one nucleic acid construct or vector or in severalones. Advantageously, only one copy of the nucleic acid molecule of theinvention or its encoding genes is present in the nucleic acid constructor vector. Several vectors or nucleic acid construct or vector can beexpressed together in the host organism. The nucleic acid molecule orthe nucleic acid construct or vector according to the invention can beinserted in a vector and be present in the cell in a free form. If astable transformation is preferred, a vector is used, which is stablyduplicated over several generations or which is else be inserted intothe genome. In the case of plants, integration into the plastid genomeor, in particular, into the nuclear genome may have taken place. For theinsertion of more than one gene in the host genome the genes to beexpressed are present together in one gene construct, for example inabove-described vectors bearing a plurality of genes.

As a rule, regulatory sequences for the expression rate of a gene arelocated upstream (5′), within, and/or downstream (3′) relative to to thecoding sequence of the nucleic acid molecule of the invention or anothercodogenic gene segment. They control in particular transcription and/ortranslation and/or the transcript stability. The expression level isdependent on the conjunction of further cellular regulatory systems,such as the protein biosynthesis and degradation systems of the cell.

Regulatory sequences include transcription and translation regulatingsequences or signals, e.g. sequences located upstream (5′), whichconcern in particular the regulation of transcription or translationinitiation, such as promoters or start codons, and sequences locateddownstream (3′), which concern in particular the regulation oftranscription or translation termination and transcript stability, suchas polyadenylation signals or stop codons. Regulatory sequences can alsobe present in transcribed coding regions as well in transcribednon-coding regions, e.g. in introns, as for example splicing sites.Promoters for the regulation of expression of the nucleic acid moleculeaccording to the invention in a cell and which can be employed are, inprinciple, all those which are capable of stimulating the transcriptionof genes in the organisms in question, such as microorganisms or plants.Suitable promoters, which are functional in these organisms, aregenerally known. They may take the form of constitutive or induciblepromoters. Suitable promoters can enable the development- and/ortissue-specific expression in multi-celled eukaryotes; thus, leaf-,root-, flower-, seed-, stomata-, tuber- or fruit-specific promoters mayadvantageously be used in plants.

The regulatory sequences or factors can, as described above, have apositive effect on, the expression of the genes introduced, thusincreasing their expression. Thus, an enhancement of the expression canadvantageously take place at the transcriptional level by using strongtranscription signals such as strong promoters and/or strong enhancers.In addition, enhancement of expression on the translational level isalso possible, for example by introducing translation enhancersequences, e.g., the Ω enhancer e.g. improving the ribosomal binding tothe transcript, or by increasing the stability of the mRNA, e.g. byreplacing the 3′UTR coding region by a region encoding a 3′UTR known asconferring an high stability of the transcript or by stabilization ofthe transcript through the elimination of transcript instability, sothat the mRNA molecule is translated more often than the wild type. Forexample in plants AU-rich elements (AREs) and DST (downstream) elementsdestabilized transcripts. Mutagenesis studies have demonstrated thatresidues within two of the conserved domains, the ATAGAT and the GTAregions, are necessary for instability function. Therefore removal ormutation of such elements would obviously lead to more stabletranscripts, higher transcript rates and higher protein acitivity.Translation enhancers are also the “overdrive sequence”, which comprisesthe tobacco mosaic virus 5′-untranslated leader sequence and whichincreases the protein/RNA ratio (Gallie et al., 1987, Nucl. AcidsResearch 15:8693-8711)

Enhancers are generally defined as cis active elements, which canstimulate gene transcription independent of position and orientation.Different enhancers have been identified in plants, which can eitherstimulate transcription constitutively or tissue or stimuli specific.Well known examples for constitutive enhancers are the enhancer from the35S promoter (Odell et al., 1985, Nature 313:810-812) or the ocsenhancer (Fromm et al., 1989, Plant Cell 1: 977:984) Another examplesare the G-Box motif tetramer which confers high-level constitutiveexpression in dicot and monocot plants (Ishige et al., 1999, PlantJournal, 18, 443-448) or the petE, a A/T-rich sequence which act asquantitative enhancers of gene expression in transgenic tobacco andpotato plants (Sandhu et al., 1998; Plant Mol Biol. 37(5):885-96).Beside that, a large variety of cis-active elements have been describedwhich contribute to specific expression pattern, like organ specificexpression or induced expression in response to biotic or abioticstress. Examples are elements, which provide pathogen or wound-inducedexpression (Rushton, 2002, Plant Cell, 14, 749-762) or guardcell-specific expression (Plesch, 2001, Plant Journal 28, 455-464).

Advantageous regulatory sequences for the expression of the nucleic acidmolecule according to the invention in microorganisms are present forexample in promoters such as the cos, tac, rha, trp, tet, trp-tet, lpp,lac, lpp-lac, lacl^(q−), T7, T5, T3, gal, trc, ara, SP6, λ-P_(R) orλ-P_(L) promoter, which are advantageously used in Gram-negativebacteria. Further advantageous regulatory sequences are present forexample in the Gram-positive promoters amy, dnak, xylS and SPO2, in theyeast or fungal promoters ADC1, MFα, AC, P-60, UASH, MCB, PHO, CYC1,GAPDH, TEF, rp28, ADH. Promoters, which are particularly advantageous,are constitutive, tissue or compartment specific and induciblepromoters. In general, “promoter” is understood as meaning, in thepresent context, a regulatory sequence in a nucleic acid molecule, whichmediates the expression of a coding sequence segment of a nucleic acidmolecule. In general, the promoter is located upstream to the codingsequence segment. Some elements, for example expression-enhancingelements such as enhancer may, however, also be located downstream oreven in the transcribed region.

In principle, it is possible to use natural promoters together withtheir regulatory sequences, such as those mentioned above, for the novelprocess. It is also possible advantageously to use synthetic promoters,either additionally or alone, in particular when they mediateseed-specific expression such as described in, for example, WO 99/16890.

The expression of the nucleic acid molecules used in the process may bedesired alone or in combination with other genes or nucleic acids.Multiple nucleic acid molecules conferring the expression ofadvantageous genes can be introduced via the simultaneous transformationof several individual suitable nucleic acid constructs, i.e. expressionconstructs, or, preferably, by combining several expression cassettes onone construct. It is also possible to transform several vectors with ineach case several expression cassettes stepwise into the recipientorganisms.

As described above, the transcription of the genes introduced shouldadvantageously be terminated by suitable terminators at the 3′ end ofthe biosynthesis genes introduced (behind the stop codon). A terminator,which may be used for this purpose is, for example, the OCS1 terminator,the nos3 terminator or the 35S terminator. As is the case with thepromoters, different terminator sequences should be used for each gene.Terminators, which are useful in microorganism, are for example the fimAterminator, txn terminator or trp terminator. Such terminators can berho-dependent or rho-independent.

Different plant promoters such as, for example, the USP, the LegB4-, theDC3 promoter or the ubiquitin promoter from parsley or other hereinmentioned promoter and different terminators may advantageously be usedin the nucleic acid construct.

In order to ensure the stable integration, into the transgenic plant, ofnucleic acid molecules used in the process according to the invention incombination with further biosynthesis genes over a plurality ofgenerations, each of the coding regions used in the process should beexpressed under the control of its own, preferably unique, promotersince repeating sequence motifs may lead to recombination events or tosilencing or, in plants, to instability of the T-DNA.

The nucleic acid construct is advantageously constructed in such a waythat a promoter is followed by a suitable cleavage site for insertion ofthe nucleic acid to be expressed, advantageously in a polylinker,followed, if appropriate, by a terminator located behind the polylinker.If appropriate, this order is repeated several times so that severalgenes are combined in one construct and thus can be introduced into thetransgenic plant in order to be expressed. The sequence isadvantageously repeated up to three times. For the expression, thenucleic acid sequences are inserted via the suitable cleavage site, forexample in the polylinker behind the promoter. It is advantageous foreach nucleic acid sequence to have its own promoter and, if appropriate,its own terminator, as mentioned above. However, it is also possible toinsert several nucleic acid sequences behind a promoter and, ifappropriate, before a terminator if a polycistronic transcription ispossible in the host or target cells. In this context, the insertionsite, or the sequence of the nucleic acid molecules inserted, in thenucleic acid construct is not decisive, that is to say a nucleic acidmolecule can be inserted in the first or last position in the cassettewithout this having a substantial effect on the expression. However, itis also possible to use only one promoter type in the construct.However, this may lead to undesired recombination events or silencingeffects, as said.

Accordingly, in a preferred embodiment, the nucleic acid constructaccording to the invention confers expression of the nucleic acidmolecule of the invention, and, optionally further genes, in a plant andcomprises one or more plant regulatory elements. Said nucleic acidconstruct according to the invention advantageously encompasses a plantpromoter or a plant terminator or a plant promoter and a plantterminator.

A “plant” promoter comprises regulatory elements, which mediate theexpression of a coding sequence segment in plant cells. Accordingly, aplant promoter need not be of plant origin, but may originate fromviruses or microorganisms, in particular for example from viruses whichattack plant cells.

The plant promoter can also originates from a plant cell, e.g. from theplant, which is transformed with the nucleic acid construct or vector asdescribed herein. This also applies to other “plant” regulatory signals,for example in “plant” terminators.

A nucleic acid construct suitable for plant expression preferablycomprises regulatory elements which are capable of controlling theexpression of genes in plant cells and which are operably linked so thateach sequence can fulfill its function. Accordingly, the nucleic acidconstruct can also comprise transcription terminators. Examples fortranscriptional termination are polyadenylation signals. Preferredpolyadenylation signals are those which originate from Agrobacteriumtumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACH5, whichis known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835 etseq.) or functional equivalents thereof, but all the other terminatorswhich are functionally active in plants are also suitable.

The nucleic acid construct suitable for plant expression preferably alsocomprises other operably linked regulatory elements such as translationenhancers, for example the overdrive sequence, which comprises thetobacco mosaic virus 5′-untranslated leader sequence, which increasesthe protein/RNA ratio (Gallie et al., 1987, Nucl. Acids Research15:8693-8711).

Other preferred sequences for use in operable linkage in gene expressionconstructs are targeting sequences, which are required for targeting thegene product into specific cell compartments (for a review, see Kermode,Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references citedtherein), for example into the vacuole, the nucleus, all types ofplastids, such as amyloplasts, chloroplasts, chromoplasts, theextracellular space, the mitochondria, the endoplasmic reticulum,elaioplasts, peroxisomes, glycosomes, and other compartments of cells orextracellular. Sequences, which must be mentioned in this context are,in particular, the signal-peptide- or transit-peptide-encoding sequenceswhich are known per se. For example, plastid-transit-peptide-encodingsequences enable the targeting of the expression product into theplastids of a plant cell. Targeting sequences are also known foreukaryotic and to a lower extent for prokaryotic organisms and canadvantageously be operable linked with the nucleic acid molecule of thepresent invention to achieve an expression in one of said compartmentsor extracellular.

For expression in plants, the nucleic acid molecule must, as describedabove, be linked operably to or comprise a suitable promoter whichexpresses the gene at the right point in time and in a cell- ortissue-specific manner. Usable promoters are constitutive promoters(Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those whichoriginate from plant viruses, such as 35S CAMV (Franck et al., Cell 21(1980) 285-294), 19S CaMV (see also U.S. Pat. No. 5,352,605 and WO84/02913), 34S FMV (Sanger et al., Plant. Mol. Biol., 14, 1990:433-443), the parsley ubiquitin promoter, or plant promoters such as theRubisco small subunit promoter described in U.S. Pat. No. 4,962,028 orthe plant promoters PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)],SSU, PGEL1, OCS [Leisner (1988) Proc Natl Acad Sci USA 85(5):2553-2557], lib4, usp, mas [Comai (1990) Plant Mol Biol 15 (3):373-381],STLS1, ScBV (Schenk (1999) Plant Mol Biol 39(6):1221-1230), B33, SAD1 orSAD2 (flax promoters, Jain et al., Crop Science, 39 (6), 1999:1696-1701) or nos [Shaw et al. (1984) Nucleic Acids Res.12(20):7831-7846]. Stable, constitutive expression of the proteinsaccording to the invention a plant can be advantageous. However,inducible expression of the polypeptide of the invention isadvantageous, if a late expression before the harvest is of advantage,as metabolic manipulation may lead to a plant growth retardation.

The expression of plant genes can also be facilitated as described abovevia a chemical inducible promoter (for a review, see Gatz 1997, Annu.Rev. Plant Physiol. Plant Mol. Biol., 48:89-108). Chemically induciblepromoters are particularly suitable when it is desired to express thegene in a time-specific manner. Examples of such promoters are asalicylic acid inducible promoter (WO 95/19443), and abscisicacid-inducible promoter (EP 335 528), a tetracyclin-inducible promoter(Gatz et al. (1992) Plant J. 2, 397-404), a cyclohexanol- orethanol-inducible promoter (WO 93/21334) or others as described herein.

Other suitable promoters are those which react to biotic or abioticstress conditions, for example the pathogen-induced PRP1 gene promoter(Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the tomatoheat-inducible hsp80 promoter (U.S. Pat. No. 5,187,267), the potatochill-inducible alpha-amylase promoter (WO 96/12814) or thewound-inducible pinII promoter (EP-A-0 375 091) or others as describedherein.

Preferred promoters are in particular those which bring about geneexpression in tissues and organs in which the biosynthesis of finechemical takes place, in seed cells, such as endosperm cells and cellsof the developing embryo. Suitable promoters are the oilseed rape napingene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USP promoter(Baeumlein et al., Mol Gen Genet, 1991, 225 (3):459-67), the Arabidopsisoleosin promoter (WO 98/45461), the Phaseolus vulgaris phaseolinpromoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter (WO91/13980), the bean arc5 promoter, the carrot DcG3 promoter, or theLegumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2(2):233-9), and promoters which bring about the seed-specific expressionin monocotyledonous plants such as maize, barley, wheat, rye, rice andthe like. Advantageous seed-specific promoters are the sucrose bindingprotein promoter (WO 00/26388), the phaseolin promoter and the napinpromoter. Suitable promoters which must be considered are the barleylpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230), and thepromoters described in WO 99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the wheat glutelin gene, the maize zeingene, the oat glutelin gene, the sorghum kasirin gene and the ryesecalin gene). Further suitable promoters are Amy32b, Amy 6-6 andAleurain [U.S. Pat. No. 5,677,474], Bce4 (oilseed rape) [U.S. Pat. No.5,530,149], glycinin (soya) [EP 571 741], phosphoenolpyruvatecarboxylase (soya) [JP 06/62870], ADR12-2 (soya) [WO 98/08962],isocitrate lyase (oilseed rape) [U.S. Pat. No. 5,689,040] or α-amylase(barley) [EP 781 849]. Other promoters which are available for theexpression of genes in plants are leaf-specific promoters such as thosedescribed in DE-A 19644478 or light-regulated promoters such as, forexample, the pea petE promoter.

Further suitable plant promoters are the cytosolic FBPase promoter orthe potato ST-LSI promoter (Stockhaus et al., EMBO J. 8, 1989, 2445),the Glycine max phosphoribosylpyrophosphate amidotransferase promoter(GenBank Accession No. U87999) or the node-specific promoter describedin EP-A-0 249 676.

Other promoters, which are particularly suitable, are those, which bringabout plastid-specific expression. Suitable promoters such as the viralRNA polymerase promoter are described in WO 95/16783 and WO 97/06250,and the Arabidopsis clpP promoter, which is described in WO 99/46394.

Other promoters, which are used for the strong expression ofheterologous sequences in as many tissues as possible, in particularalso in leaves, are, in addition to several of the abovementioned viraland bacterial promoters, preferably, plant promoters of actin orubiquitin genes such as, for example, the rice actin1 promoter. Furtherexamples of constitutive plant promoters are the sugarbeet V-ATPasepromoters (WO 01/14572). Examples of synthetic constitutive promotersare the Super promoter (WO 95/14098) and promoters derived from G-boxes(WO 94/12015). If appropriate, chemical inducible promoters mayfurthermore also be used, compare EP-A 388186, EP-A 335528, WO 97/06268.

As already mentioned herein, further regulatory sequences, which may beexpedient, if appropriate, also include sequences, which target thetransport and/or the localization of the expression products. Sequences,which must be mentioned in this context are, in particular, thesignal-peptide- or transit-peptide-encoding sequences which are knownper se. For example, plastid-transit-peptide-encoding sequences enablethe targeting of the expression product into the plastids of a plantcell.

Preferred recipient plants are, as described above, in particular thoseplants, which can be transformed in a suitable manner. These includemonocotyledonous and dicotyledonous plants. Plants which must bementioned in particular are agriculturally useful plants such as cerealsand grasses, for example Triticum spp., Zea mays, Hordeum vulgare, oats,Secale cereale, Oryza sativa, Pennisetum glaucum, Sorghum bicolor,Triticale, Agrostis spp., Cenchrus ciliaris, Dactylis glomerata, Festucaarundinacea, Lolium spp., Medicago spp. and Saccharum spp., legumes andoil crops, for example Brassica juncea, Brassica napus, Glycine max,Arachis hypogaea, Gossypium hirsutum, Cicer arietinum, Helianthusannuus, Lens culinaris, Linum usitatissimum, Sinapis alba, Trifoliumrepens and Vicia narbonensis, vegetables and fruits, for examplebananas, grapes, Lycopersicon esculentum, asparagus, cabbage,watermelons, kiwi fruit, Solanum tuberosum, Beta vulgaris, cassava andchicory, trees, for example Coffea species, Citrus spp., Eucalyptusspp., Picea spp., Pinus spp. and Populus spp., medicinal plants andtrees, and flowers.

One embodiment of the present invention also relates to a method forgenerating a vector, which comprises the insertion, into a vector, ofthe nucleic acid molecule characterized herein, the nucleic acidmolecule according to the invention or the expression cassette accordingto the invention. The vector can, for example, be introduced in to acell, e.g. a microorganism or a plant cell, as described herein for thenucleic acid construct, or below under transformation or transfection orshown in the examples. A transient or stable transformation of the hostor target cell is possible, however, a stable transformation ispreferred. The vector according to the invention is preferably a vector,which is suitable for expressing the polypeptide according to theinvention in a plant. The method can thus also encompass one or moresteps for integrating regulatory signals into the vector, in particularsignals, which mediate the expression in microorganisms or plants.

Accordingly, the present invention also relates to a vector comprisingthe nucleic acid molecule characterized herein as part of a nucleic acidconstruct suitable for plant expression or the nucleic acid moleculeaccording to the invention.

The advantageous vectors of the invention comprise the nucleic acidmolecules which encode proteins according to the invention, nucleic acidmolecules which are used in the process, or nucleic acid constructsuitable for plant expression comprising the nucleic acid moleculesused, either alone or in combination with further genes such as thebiosynthesis or regulatory genes of the fine chemical metabolism e.g.with the genes mentioned herein above. In accordance with the invention,the term “vector” refers to a nucleic acid molecule, which is capable oftransporting another nucleic acid to which it is linked. One type ofvector is a “plasmid”, which means a circular double-stranded DNA loopinto which additional DNA segments can be ligated. A further type ofvector is a viral vector, it being possible to ligate additional nucleicacids segments into the viral genome. Certain vectors are capable ofautonomous replication in a host cell into which they have beenintroduced (for example bacterial vectors with bacterial replicationorigin). Other preferred vectors are advantageously completely or partlyintegrated into the genome of a host cell when they are introduced intothe host cell and thus replicate together with the host genome.Moreover, certain vectors are capable of controlling the expression ofgenes with which they are in operable linkage. In the present context,these vectors are referred to as “expression vectors”. As mentionedabove, they are capable of autonomous replication or may be integratedpartly or completely into the host genome. Expression vectors, which aresuitable for DNA recombination techniques usually, take the form ofplasmids. In the present description, “plasmid” and “vector” can be usedinterchangeably since the plasmid is the most frequently used form of avector. However, the invention is also intended to encompass these otherforms of expression vectors, such as viral vectors, which exert similarfunctions. The term vector is furthermore also to encompass othervectors which are known to the skilled worker, such as phages, virusessuch as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids,cosmids, and linear or circular DNA.

The recombinant expression vectors which are advantageously used in theprocess comprise the nucleic acid molecules according to the inventionor the nucleic acid construct according to the invention in a form whichis suitable for expressing, in a host cell, the nucleic acid moleculesaccording to the invention or described herein. Accordingly, the therecombinant expression vectors comprise one or more regulatory signalsselected on the basis of the host cells to be used for the expression,in operable linkage with the nucleic acid sequence to be expressed.

In a recombinant expression vector, “operable linkage” means that thenucleic acid molecule of interest is linked to the regulatory signals insuch a way that expression of the nucleic acid molecule is possible:they are linked to one another in such a way that the two sequencesfulfill the predicted function assigned to the sequence (for example inan in-vitro transcription/translation system, or in a host cell if thevector is introduced into the host cell).

The term “regulatory sequence” is intended to comprise promoters,enhancers and other expression control elements (for examplepolyadenylation signals. These regulatory sequences are described, forexample, in Goeddel: Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990), or see: Gruber andCrosby, in: Methods in Plant Molecular Biology and Biotechnolgy, CRCPress, Boca Raton, Fla., Ed.: Glick and Thompson, chapter 7, 89-108,including the references cited therein. Regulatory sequences encompassthose, which control the constitutive expression of a nucleotidesequence in many types of host cells and those which control the directexpression of the nucleotide sequence in specific host cells only, andunder specific conditions. The skilled worker knows that the design ofthe expression vector may depend on factors such as the selection of thehost cell to be transformed, the extent to which the desired protein isexpressed, and the like. A preferred selection of regulatory sequencesis described above, for example promoters, terminators, enhancers andthe like. The term regulatory sequence is to be considered as beingencompassed by the term regulatory signal. Several advantageousregulatory sequences, in particular promoters and terminators aredescribed above. In general, the regulatory sequences described asadvantageous for nucleic acid construct suitable for expression are alsoapplicable for vectors.

The recombinant expression vectors used can be designed specifically forthe expression, in prokaryotic and/or eukaryotic cells, of nucleic acidmolecules used in the process. This is advantageous since intermediatesteps of the vector construction are frequently carried out inmicroorganisms for the sake of simplicity. For example, the genesaccording to the invention and other genes can be expressed in bacterialcells, insect cells (using baculovirus expression vectors), yeast cellsand other fungal cells [Romanos (1992), Yeast 8:423-488; van den Hondel,(1991), in: More Gene Manipulations in Fungi, J. W. Bennet & L. L.Lasure, Ed., pp. 396-428: Academic Press: San Diego; and van den Hondel,C. A. M. J. J. (1991), in: Applied Molecular Genetics of Fungi, Peberdy,J. F., et al., Ed., pp. 1-28, Cambridge University Press: Cambridge],algae [Falciatore et al., 1999, Marine Biotechnology. 1, 3:239-251]using vectors and following a transformation method as described in WO98/01572, and preferably in cells of multi-celled plants [see Schmidt,R. and Willmitzer, L. (1988) Plant Cell Rep.: 583-586; Plant MolecularBiology and Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, pp.71-119 (1993); F. F. White, in: Transgenic Plants, Bd. 1, Engineeringand Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-43;Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991),205-225 (and references cited therein)]. Suitable host cells arefurthermore discussed in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). As analternative, the sequence of the recombinant expression vector can betranscribed and translated in vitro, for example using T7promotor-regulatory sequences and T7 polymerase.

Proteins can be expressed in prokaryotes using vectors comprisingconstitutive or inducible promoters, which control the expression offusion proteins or nonfusion proteins. Typical fusion expression vectorsare, inter alia, pGEX (Pharmacia Biotech Inc; Smith, D. B., and Johnson,K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.)and pRIT5 (Pharmacia, Piscataway, N.J.), in whichglutathione-S-transferase (GST), maltose-E-binding protein or protein Ais fused with the recombinant target protein. Examples of suitableinducible nonfusion E. coli expression vectors are, inter alia, pTrc(Amann et al. (1988) Gene 69:301-315) and pET 11d [Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 60-89]. The target gene expression of the pTrcvector is based on the transcription of a hybrid trp-lac fusion promoterby the host RNA polymerase. The target gene expression from the pET 11dvector is based on the transcription of a T7-gn10-lac fusion promoter,which is mediated by a coexpressed viral RNA polymerase (T7 gn1). Thisviral polymerase is provided by the host strains BL21 (DE3) or HMS174(DE3) by a resident λ-prophage, which harbors a T7 gn1 gene under thetranscriptional control of the lacUV 5 promoter.

Other vectors which are suitable in prokaryotic organisms are known tothe skilled worker; these vectors are for example in E. coli pLG338,pACYC184, the pBR series, such as pBR322, the pUC series such as pUC18or pUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24,pLG200, pUR290, pIN-III¹¹³-B1, λgt11 or pBdCl, in Streptomyces pIJ101,pIJ364, pIJ702 or pIJ361, in Bacillus pUB 110, pC194 or pBD214, inCorynebacterium pSA77 or pAJ667.

In a further embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in the yeasts S. cerevisiaeencompass pYeDesaturasec1 (Baldari et al. (1987) Embo J. 6:229-234),pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz etal. (1987) Gene 54:113-123) and pYES2 (Invitrogen Corporation, SanDiego, Calif.). Vectors and methods for the construction of vectorswhich are suitable for use in other fungi, such as the filamentousfungi, encompass those which are described in detail in: van den Hondel,C. A. M. J. J. [(1991), J. F. Peberdy, Ed., pp. 1-28, CambridgeUniversity Press: Cambridge; or in: More Gene Manipulations in Fungi; J.W. Bennet & L. L. Lasure, Ed., pp. 396-428: Academic Press: San Diego].Examples of other suitable yeast vectors are 20□M, pAG-1, YEp6, YEp13 orpEMBLYe23.

Further vectors, which may be mentioned by way of example, are pALS1,pIL2 or pBB116 in fungi or pLGV23, pGHlac⁺, pBIN19, pAK2004 or pDH51 inplants.

As an alternative, the nucleic acid sequences can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectors whichare available for expressing proteins in cultured insect cells (forexample Sf9 cells) encompass the pAc series (Smith et al. (1983) Mol.Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989)Virology 170:31-39).

The abovementioned vectors are only a small overview of potentiallysuitable vectors. Further plasmids are known to the skilled worker andare described, for example, in: Cloning Vectors (Ed. Pouwels, P. H., etal., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).Further suitable expression systems for prokaryotic and eukaryoticcells, see the chapters 16 and 17 by Sambrook, J., Fritsch, E. F., andManiatis, T., Molecular Cloning: A Laboratory Manual, 2nd Edition, ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Accordingly, one embodiment of the invention relates to a vector wherethe nucleic acid molecule according to the invention is linked operablyto regulatory sequences which permit the expression in a prokaryotic oreukaryotic or in a prokaryotic and eukaryotic host.

Accordingly, one embodiment of the invention relates to a host cell,which has been transformed stably or transiently with the vectoraccording to the invention or the nucleic acid molecule according to theinvention or the nucleic acid construct according to the invention.

Depending on the host organism, the organisms used in the processaccording to the invention are cultured or grown in a manner with whichthe skilled worker is familiar. As a rule, microorganisms are grown in aliquid medium comprising a carbon source, usually in the form of sugars,a nitrogen source, usually in the form of organic nitrogen sources suchas yeast extract or salts such as ammonium sulfate, trace elements suchas iron salts, manganese salts, magnesium salts, and, if appropriate,vitamins, at temperatures between 0° C. and 100° C., preferably between10° C. and 60° C., while passing in oxygen. In the event themicroorganism is anaerobe, no oxygen is blown through the culturemedium. The pH value of the liquid nutrient medium may be kept constant,that is to say regulated during the culturing phase, or not. Theorganisms may be cultured batchwise, semibatchwise or continuously.Nutrients may be provided at the beginning of the fermentation or fed insemicontinuously or continuously.

The fine chemical produced can be isolated from the organism by methodswith which the skilled worker is familiar. For example via extraction,salt precipitation and/or ion-exchange chromatography etc. To this end,the organisms may be disrupted beforehand. The process according to theinvention can be conducted batchwise, semibatchwise or continuously. Asummary of known culture and isolation techniques can be found in thetextbook by Chmiel [Bioprozeβtechnik 1, Einführung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)], Demainet al. (Industrial Microbiology and Biotechnology, second edition, ASMPress, Washington, D.C., 1999, ISBN 1-55581-128-0] or in the textbook byStorhas [Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)].

In one embodiment, the present invention relates to a polypeptideencoded by the nucleic acid molecule according to the present invention,preferably conferring an increase in the fine chemical content in anorganism or cell after increasing the expression or activity.

The present invention also relates to a process for the production of apolypeptide according to the present invention, the polypeptide beingexpressed in a host cell according to the invention, preferably in amicroorganism or a transgenic plant cell.

In one embodiment, the nucleic acid molecule used in the process for theproduction of the polypeptide is derived from a microorganism,preferably from a prokaryotic or protozoic cell with a eukaryoticorganism as host cell. E.g., in one embodiment the polypeptide isproduced in a plant cell or plant with a nucleic acid molecule derivedfrom a prokaryote or a fungus or an alga or another microorganismus butnot from plant.

The skilled worker knows that protein and DNA expressed in differentorganisms differ in many respects and properties, e.g. DNA modulationand imprinting, such as methylation or post-translational modification,as for example glucosylation, phosphorylation, acetylation,myristoylation, ADP-ribosylation, farnesylation, carboxylation,sulfation, ubiquination, etc. though having the same coding sequence.Preferably, the cellular expression control of the corresponding proteindiffers accordingly in the control mechanisms controlling the activityand expression of an endogenous protein or another eukaryotic protein.One major difference between proteins expressed in prokaryotic oreukaryotic organisms is the amount and pattern of glycosylation. Forexample in E. coli there are no glycosylated proteins. Proteinsexpressed in yeasts have high mannose content in the glycosylatedproteins, whereas in plants the glycosylation pattern is complex.

The polypeptide of the present invention is preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the protein is cloned into a vector (as described above), thevector is introduced into a host cell (as described above) and saidpolypeptide is expressed in the host cell. Said polypeptide can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Alternative to recombinantexpression, the polypeptide or peptide of the present invention can besynthesized chemically using standard peptide synthesis techniques.

Moreover, native polypeptides conferring the increase of the finechemical in an organism or part thereof can be isolated from cells(e.g., endothelial cells), for example using the antibody of the presentinvention as described below, in particular, an anti-YNL090W proteinantibody or an antibody against polypeptides as depicted in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282,284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394,which can be produced by standard techniques utilizing the polypeptid ofthe present invention or fragment thereof, i.e., the polypeptide of thisinvention. Preferred are monoclonal antibodies.

In one embodiment, the present invention relates to a polypeptide havingthe amino acid sequence encoded by a nucleic acid molecule of theinvention or obtainable by a process of the invention. Said polypeptideconfers preferably the aforementioned activity, in particular, thepolypeptide confers the increase of the fine chemical in a cell or anorgansim or a part thereof after increasing the cellular activity, e.g.by increasing the expression or the specific activity of thepolypeptide.

In one embodiment, the present invention relates to a polypeptide havingthe sequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392 or 394 or as coded by the nucleic acidmolecule as depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295,297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351,353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379,381, 383, 385, 387, 389, 391or 393 or functional homologues thereof.

In one advantageous embodiment, in the method of the present inventionthe activity of a polypeptide is increased comprising or consisting ofthe consensus sequence as depicted in SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 397,SEQ ID NO: 398, SEQ ID NO: 399 and/or SEQ ID NO: 400 and in one anotherembodiment, the present invention relates to a polypeptide comprising orconsisting of the consensus sequence as depicted in SEQ ID NO: 47, SEQID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399 and/or SEQ ID NO: 400whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6, morepreferred 5 or 4, even more preferred 3, even more preferred 2, evenmore preferred 1, most preferred 0 of the amino acids positionsindicated can be replaced by any amino acid.

In one embodiment not more than 15%, preferably 10%, even more preferred5%, 4%, 3%, or 2%, most preferred 1% or 0% of the amino acid positionindicated by a letter are/is replaced another amino acid.

In one embodiment 20 or less, preferably 15 or 10, preferably 9, 8, 7,or 6, more preferred 5 or 4, even more preferred 3, even more preferred2, even more preferred 1, most preferred 0 amino acids are inserted intothe consensus sequence.

The consensus sequences of specified domains were derived from amultiple alignment of all sequences. The consensus sequences aredisclosed under SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 397, SEQ ID NO: 398, SEQ IDNO: 399 and/or SEQ ID NO: 400. The letters represent the three letteramino acid code and indicate that the amino acids are conserved in allaligned proteins. The letter Xaa stands for amino acids, which are notconserved in all sequences. In some cases of the sequences as depictedin SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ IDNO: 51, SEQ ID NO: 52, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399and/or SEQ ID NO: 400 preferred amino acids are mentioned in thesequence protocol in others all natural amino acids are possible. FIG. 1shows an alignment with part of the sequences in the one letter aminoacid code. The conserved sequences are framed.

The alignment was performed either with the Software AlignX (Sep. 25,2002) a component of Vector NTI Suite 8.0, InforMax™, Invitrogen™ lifescience software, U.S. Main Office, 7305 Executive Way, Frederick, Md.21704, USA with the following settings: For pairwise alignments: gapopening penality: 10.0; gap extension penality 0.1. For multiplealignments: Gap opening penalty: 10.0; Gap extension penalty: 0.1; Gapseparation penalty range: 8; Residue substitution matrix: blosum62;Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0.5;Consensus calculation options: Residue fraction for consensus: 1 orpreferably the percent sequence identity between two nucleic acid orpolypeptide sequences was determined using the Vector NTI 6.0 (PC)software package (InforMax, 7600 Wisconsin Ave., Bethesda, Md. 20814). Agap opening penalty of 15 and a gap extension penalty of 6.66 are usedfor determining the percent identity of two nucleic acids. A gap openingpenalty of 10 and a gap extension penalty of 0.1 are used fordetermining the percent identity of two polypeptides. All otherparameters are set at the default settings. For purposes of a multiplealignment (Clustal W algorithm), the gap opening penalty is 10, and thegap extension penalty is 0.05 with blosum62 matrix.

In one advantageous embodiment, the method of the present inventioncomprises the increasing of a polypeptide comprising or consisting ofplant or microorganism specific consensus sequences.

In one embodiment, said polypeptide of the invention distinguishes overthe sequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392 or 394 by one or more amino acids. Inone embodiment, polypeptide distinguishes form the sequence as depictedin SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394 by more than 5, 6, 7, 8 or 9 amino acids, preferably bymore than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are morethan 40, 50, or 60 amino acids and, preferably, the sequence of thepolypeptide of the invention distinguishes from the sequence as depictedin SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394 by not more than 80% or 70% of the amino acids,preferably not more than 60% or 50%, more preferred not more than 40% or30%, even more preferred not more than 20% or 10%. In an otherembodiment, said polypeptide of the invention does not consist of thesequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392 or 394.

In one embodiment, the polypeptide of the invention comprises any one ofthe sequences not known to the public before. In one embodiment, thepolypeptide of the invention orginates from a non-plant cell, inparticular from a microorganism, and was expressed in a plant cell. Inone embodiment, the present invention relates to a polypeptide encodedby the nucleic acid molecule of the invention or used in the process ofthe invention for which an activity has not been described yet.

In one embodiment, the invention relates to polypeptide conferring anincrease in the fine chemical in an organism or part being encoded bythe nucleic acid molecule of the invention or used in the process of theinvention and having a sequence which distinguishes from the sequence asdepicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300,302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328,330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,386, 388, 390, 392 or 394 by one or more amino acids. In an otherembodiment, said polypeptide of the invention does not consist of thesequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392 or 394. In a further embodiment, saidpolypeptide of the present invention is less than 100%, 99.999%, 99.99%,99.9% or 99% identical. In one embodiment, said polypeptide does notconsist of the sequence encoded by the nucleic acid molecules asdepicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327,329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,385, 387, 389, 391 or 393.

In one embodiment, the present invention relates to a polypeptide havingthe biological activity represented by a protein as depicted in SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280,282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308,310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336,338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or394 and which distinguishes over the aforementioned sequences by one ormore amino acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,preferably by more than 10, 15, 20, 25 or 30 amino acids, evenmorepreferred are more than 40, 50, or 60 amino acids but even morepreferred by less than 70% of the amino acids, more preferred by lessthan 50%, even more preferred my less than 30% or 25%, more preferredare 20% or 15%, even more preferred are less than 10%.

The terms “protein” and “polypeptide” used in this application areinterchangeable. “Polypeptide” refers to a polymer of amino acids (aminoacid sequence) and does not refer to a specific length of the molecule.Thus peptides and oligopeptides are included within the definition ofpolypeptide. This term does also refer to or include post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),polypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.

Preferably, the polypeptide is isolated. An “isolated” or “purified”protein or nucleic acid molecule or biologically active portion thereofis substantially free of cellular material when produced by recombinantDNA techniques or chemical precursors or other chemicals when chemicallysynthesized.

The language “substantially free of cellular material” includespreparations of the polypeptide of the invention in which the protein isseparated from cellular components of the cells in which it is naturallyor recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations havingless than about 30% (by dry weight) of “contaminating protein”, morepreferably less than about 20% of “contaminating protein”, still morepreferably less than about 10% of “contaminating protein”, and mostpreferably less than about 5% “contaminating protein”. The term“Contaminating protein” relates to polypeptides, which are notpolypeptides of the present invention. When the polypeptide of thepresent invention or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation. The language “substantiallyfree of chemical precursors or other chemicals” includes preparations inwhich the polypeptide of the present invention is separated fromchemical precursors or other chemicals, which are involved in thesynthesis of the protein. The language “substantially free of chemicalprecursors or other chemicals” includes preparations having less thanabout 30% (by dry weight) of chemical precursors or non-polypeptide ofthe inventionchemicals, more preferably less than about 20% chemicalprecursors or non-polypeptide of the inventionchemicals, still morepreferably less than about 10% chemical precursors or non-polypeptide ofthe invention-chemicals, and most preferably less than about 5% chemicalprecursors or non-polypeptide of the invention-chemicals. In preferredembodiments, isolated proteins or biologically active portions thereoflack contaminating proteins from the same organism from which thepolypeptide of the present invention is derived. Typically, suchproteins are produced by recombinant techniques.

A polypeptide of the invention can participate in the process of thepresent invention. The polypeptide or a portion thereof comprisespreferably an amino acid sequence which is sufficiently homologous to anamino acid sequence as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392 or 394 such that the protein orportion thereof maintains the ability to confer the activity of thepresent invention. The portion of the protein is preferably abiologically active portion as described herein. Preferably, thepolypeptide used in the process of the invention has an amino acidsequence identical as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392 or 394.

Further, the polypeptide can have an amino acid sequence which isencoded by a nucleotide sequence which hybridizes, preferably hybridizesunder stringent conditions as described above, to a nucleotide sequenceof the nucleic acid molecule of the present invention. Accordingly, thepolypeptide has an amino acid sequence which is encoded by a nucleotidesequence that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or70%, preferably at least about 75%, 80%, 85% or 90, and more preferablyat least about 91%, 92%, 93%, 94% or 95%, and even more preferably atleast about 96%, 97%, 98%, 99% or more homologous to one of the aminoacid sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326,328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,384, 386, 388, 390, 392 or 394. The preferred polypeptide of the presentinvention preferably possesses at least one of the activities accordingto the invention and described herein. A preferred polypeptide of thepresent invention includes an amino acid sequence encoded by anucleotide sequence which hybridizes, preferably hybridizes understringent conditions, to a nucleotide sequence of SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393 or which ishomologous thereto, as defined above.

Accordingly the polypeptide of the present invention can vary from SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306,308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334,336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362,364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,392 or 394 in amino acid sequence due to natural variation ormutagenesis, as described in detail herein. Accordingly, the polypeptidecomprise an amino acid sequence which is at least about 35%, 40%, 45%,50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or90, and more preferably at least about 91%, 92%, 93%, 94% or 95%, andmost preferably at least about 96%, 97%, 98%, 99% or more homologous toan entire amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392 or 394.

For the comparison of amino acid sequences the same algorithms asdescribed above or nucleic acid sequences can be used. Results of highquality are reached by using the algorithm of Needleman and Wunsch orSmith and Waterman. Therefore programs based on said algorithms arepreferred. Advantageously the comparisons of sequences can be done withthe program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins etal., CABIOS, 5 1989: 151-153) or preferably with the programs Gap andBestFit, which are respectively based on the algorithms of Needleman andWunsch [J. Mol. Biol. 48; 443-453 (1970)] and Smith and Waterman [Adv.Appl. Math. 2; 482-489 (1981)]. Both programs are part of the GCGsoftware-package [Genetics Computer Group, 575 Science Drive, Madison,Wis., USA 53711 (1991); Altschul et al. (1997) Nucleic Acids Res.25:3389 et seq.]. Therefore preferably the calculations to determine theperentages of sequence homology are done with the program Gap over thewhole range of the sequences. The following standard adjustments for thecomparison of amino acid sequences were used: gap weight: 8, lengthweight: 2, average match: 2.912, average mismatch: −2.003.

Biologically active portions of an polypeptide of the present inventioninclude peptides comprising amino acid sequences derived from the aminoacid sequence of the polypeptide of the present invention or used in theprocess of the present invention, e.g., the amino acid sequence shown inSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276,278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392 or 394 or the amino acid sequence of a protein homologousthereto, which include fewer amino acids than a full length polypeptideof the present invention or used in the process of the present inventionor the full length protein which is homologous to an polypeptide of thepresent invention or used in the process of the present inventiondepicted herein, and exhibit at least one activity of polypeptide of thepresent invention or used in the process of the present invention.

Typically, biologically (or immunologically) active portions i.e.peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35,36, 37, 38, 39, 40, 50, 100 or more amino acids in length comprise adomain or motif with at least one activity or epitope of a polypeptideof the present invention or used in the process of the presentinvention. Moreover, other biologically active portions, in which otherregions of the polypeptide are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the activities describedherein.

Manipulation of the nucleic acid molecule of the invention may result inthe production of proteins having the biological activity represented bya protein as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392 or 394 and having differences from saidaforementioned wild-type proteins. These proteins may be improved inefficiency or activity, may be present in greater numbers in the cellthan is usual, or may be decreased in efficiency or activity in relationto the wild type protein.

Any mutagenesis strategies for the polypeptide of the present inventionor the polypeptide used in the process of the present invention toresult in increasing said activity are not meant to be limiting;variations on these strategies will be readily apparent to one skilledin the art. Using such strategies, and incorporating the mechanismsdisclosed herein, the nucleic acid molecule and polypeptide of theinvention may be utilized to generate plants or parts thereof,expressing wildtype proteins of the invention or mutated proteinencoding nucleic acid molecules and polypeptide molecules of theinvention such that the yield, production, and/or efficiency ofproduction of a desired compound is improved.

This desired compound may be any natural product of plants, whichincludes the final products of biosynthesis pathways and intermediatesof naturally-occurring metabolic pathways, as well as molecules which donot naturally occur in the metabolism of said cells, but which areproduced by a said cells of the invention. Preferrably, the compound isa composition of amino acids or a recovered amino acid, in particular,the fine chemical, free or in protein-bound form.

The invention also provides chimeric or fusion proteins.

As used herein, an “chimeric protein” or “fusion protein” comprises anpolypeptide operatively linked to a polypeptide which does not conferabove-mentioned activity, in particulare, which does not confer anincrease of content of the fine chemical in a cell or an organism or apart thereof, if its activity is increased.

In one embodiment, a protein (=polypeptide)” having the biologicalactivity represented by a protein as depicted in SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370,372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 refers to apolypeptide having an amino acid sequence corresponding to thepolypeptide of the invention or used in the process of the invention,whereas a “non-polypeptide of the invention” or “other polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous a polypeptide of theinvention, preferably which is not substantially homologous to apolypeptide having the biological activity represented by a protein asdepicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300,302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328,330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384,386, 388, 390, 392 or 394, e.g., a protein which does not confer theactivity described herein and which is derived from the same or adifferent organism.

Within the fusion protein, the term “operatively linked” is intended toindicate that the polypeptide of the invention or a polypeptide used inthe process of the invention and the “other polypeptide” or a partthereof are fused to each other so that both sequences fulfil theproposed function addicted to the sequence used. The “other polypeptide”can be fused to the N-terminus or C-terminus of the polypeptide of theinvention or used in the process of the invention. For example, in oneembodiment the fusion protein is a GST-LMRP fusion protein in which thesequences of the polypeptide of the invention or the polypeptide used inthe process of the invention are fused to the C-terminus of the GSTsequences. Such fusion proteins can facilitate the purification ofrecombinant polypeptides of the invention or a poylpeptide usefull inthe process of the invention.

In another embodiment, the fusion protein is a polypeptide of theinvention or a polypeptide used in the process of the inventioncontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofa polypeptide of the invention or a poylpeptide used in the process ofthe invention can be increased through use of a heterologous signalsequence. As already mentioned above, targeting sequences, are requiredfor targeting the gene product into specific cell compartment (for areview, see Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 andreferences cited therein), for example into the vacuole, the nucleus,all types of plastids, such as amyloplasts, chloroplasts, chromoplasts,the extracellular space, the mitochondria, the endoplasmic reticulum,elaioplasts, peroxisomes, glycosomes, and other compartments of cells orextracellular. Sequences, which must be mentioned in this context are,in particular, the signal-peptide- or transit-peptide-encoding sequenceswhich are known per se. For example, plastid-transit-peptide-encodingsequences enable the targeting of the expression product into theplastids of a plant cell. Targeting sequences are also known foreukaryotic and to a lower extent for prokaryotic organisms and canadvantageously be operable linked with the nucleic acid molecule of thepresent invention to achieve an expression in one of said compartmentsor extracellular.

Preferably, a chimeric or fusion protein of the invention is produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example by employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. The fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers, which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, for example,Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley &Sons: 1992). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST polypeptide).The nucleic acid molecule of the invention can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to theencoded protein.

Furthermore, folding simulations and computer redesign of structuralmotifs of the protein of the invention can be performed usingappropriate computer programs (Olszewski, Proteins 25 (1996), 286-299;Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679). Computer modeling ofprotein folding can be used for the conformational and energeticanalysis of detailed peptide and protein models (Monge, J. Mol. Biol.247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45).The appropriate programs can be used for the identification ofinteractive sites the polypeptide of the invention or polypeptides usedin the process of the invention and its substrates or binding factors orother interacting proteins by computer assistant searches forcomplementary peptide sequences (Fassina, Immunomethods (1994),114-120). Further appropriate computer systems for the design of proteinand peptides are described in the prior art, for example in Berry,Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci.501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The resultsobtained from the above-described computer analysis can be used for,e.g., the preparation of peptidomimetics of the protein of the inventionor fragments thereof. Such pseudopeptide analogues of the, natural aminoacid sequence of the protein may very efficiently mimic the parentprotein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224). Forexample, incorporation of easily available achiral Q-amino acid residuesinto a protein of the invention or a fragment thereof results in thesubstitution of amide bonds by polymethylene units of an aliphaticchain, thereby providing a convenient strategy for constructing apeptidomimetic (Banerjee, Biopolymers 39 (1996), 769-777).

Superactive peptidomimetic analogues of small peptide hormones in othersystems are described in the prior art (Zhang, Biochem. Biophys. Res.Commun. 224 (1996), 327-331). Appropriate peptidomimetics of the proteinof the present invention can also be identified by the synthesis ofpeptidomimetic combinatorial libraries through successive amidealkylation and testing the resulting compounds, e.g., for their bindingand immunological properties. Methods for the generation and use ofpeptidomimetic combinatorial libraries are described in the prior art,for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 andDorner, Bioorg. Med. Chem. 4 (1996), 709-715.

Furthermore, a three-dimensional and/or crystallographic structure ofthe protein of the invention can be used for the design ofpeptidomimetic inhibitors of the biological activity of the protein ofthe invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber,Bioorg. Med. Chem. 4 (1996), 1545-1558).

Furthermore, a three-dimensional and/or crystallographic structure ofthe protein of the invention and the identification of interactive sitesthe polypeptide of the invention and its substrates or binding factorscan be used for design of mutants with modulated binding or turn overactivities. For example, the active center of the polypeptide of thepresent invention can be modelled and amino acid residues participatingin the catalytic reaction can be modulated to increase or decrease thebinding of the substrate to activate or improve the polypeptide. Theidentification of the active center and the amino acids involved in thecatalytic reaction facilitates the screening for mutants having anincreased activity.

One embodiment of the invention also relates to an antibody, which bindsspecifically to the polypeptide according to the invention or parts,i.e. specific fragments or epitopes of such a protein.

The antibodies of the invention can be used to identify and isolate thepolypeptide according to the invention and encoding genes in anyorganism, preferably plants, prepared in plants described herein. Theseantibodies can be monoclonal antibodies, polyclonal antibodies orsynthetic antibodies as well as fragments of antibodies, such as Fab, Fvor scFv fragments etc. Monoclonal antibodies can be prepared, forexample, by the techniques as originally described in Köhler andMilstein, Nature 256 (1975), 495, and Galfr6, Meth. Enzymol. 73 (1981),3, which comprise the fusion of mouse myeloma cells to spleen cellsderived from immunized mammals.

Furthermore, antibodies or fragments thereof to the aforementionedpeptides can be obtained by using methods, which are described, e.g., inHarlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988. These antibodies can be used, for example, for theimmunoprecipitation and immunolocalization of proteins according to theinvention as well as for the monitoring of the synthesis of suchproteins, for example, in recombinant organisms, and for theidentification of compounds interacting with the protein according tothe invention. For example, surface plasmon resonance as employed in theBIAcore system can be used to increase the efficiency of phageantibodies selections, yielding a high increment of affinity from asingle library of phage antibodies, which bind to an epitope of theprotein of the invention (Schier, Human Antibodies Hybridomas 7 (1996),97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In many cases,the binding phenomena of antibodies to antigens are equivalent to otherligand/anti-ligand binding.

In one embodiment, the present invention relates to an antisense nucleicacid molecule comprising the complementary sequence of the nucleic acidmolecule of the present invention.

Methods to modify the expression levels and/or the activity are known topersons skilled in the art and include for instance overexpression,co-suppression, the use of ribozymes, sense and anti-sense strategies orother gene silencing approaches like RNA interference (RNAi) or promotermethylation. “Sense strand” refers to the strand of a double-strandedDNA molecule that is homologous to an mRNA transcript thereof. The“anti-sense strand” contains an inverted sequence, which iscomplementary to that of the “sense strand”.

In addition the expression levels and/or the activity can be modified bythe introduction of mutations in the regulatory or coding regions of thenucleic acids of the invention. Furthermore antibodies can be expressedwhich specifically binds to a polypeptide of interest and thereby blocksit acitivity. The protein-binding factors can, for example, also beaptamers [Famulok M and Mayer G (1999) Curr. Top Microbiol. Immunol.243:123-36] or antibodies or antibody fragments or single-chainantibodies. Obtaining these factors has been described, and the skilledworker is familiar therewith. For example, a cytoplasmic scFv antibodyhas been employed for modulating activity of the phytochrome A proteinin genetically modified tobacco plants [Owen M et al. (1992)Biotechnology (NY) 10(7): 790-794; Franken E et al. (1997) Curr. Opin.Biotechnol. 8(4): 411-416; Whitelam (1996) Trend Plant Sci. 1: 286-272].

An “antisense” nucleic acid molecule comprises a nucleotide sequence,which is complementary to a “sense” nucleic acid molecule encoding aprotein, e.g., complementary to the coding strand of a double-strandedcDNA molecule or complementary to an encoding mRNA sequence.Accordingly, an antisense nucleic acid molecule can bond via hydrogenbonds to a sense nucleic acid molecule. The antisense nucleic acidmolecule can be complementary to an entire coding strand of a nucleicacid molecule conferring the expression of the polypeptide of theinvention or used in the process of the present invention, as thenucleic acid molecule of the invention coding strand, or to only aportion thereof. Accordingly, an antisense nucleic acid molecule can beantisense to a “coding region” of the coding strand of a nucleotidesequence of a nucleic acid molecule of the present invention. The term“coding region” refers to the region of the nucleotide sequencecomprising codons, which are translated into amino acid residues.Further, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding the polypeptide of the invention or a polypeptide used in theprocess of the invention. The term “noncoding region” refers to 5′ and3′ sequences which flank the coding region that are not translated intoa polypeptide, i.e., also referred to as 5′ and 3′ untranslated regions(5′-UTR or 3′-UTR).

Given the coding strand sequences encoding the polypeptide of thepresent invention antisense nucleic acid molecules of the invention canbe designed according to the rules of Watson and Crick base pairing.

The antisense nucleic acid molecule can be complementary to the entirecoding region of the mRNA encoding the nucleic acid molecule ot theinvention or used in the process of the present invention, but can alsobe an oligonucleotide which is antisense to only a portion of the codingor noncoding region of said mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of said mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or 200nucleotides in length. An antisense nucleic acid molecule of theinvention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid molecule (e.g., an antisense oligonucleotide) canbe chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid molecule has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid molecule will be of anantisense orientation to a target nucleic acid molecule of interest,described further in the following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a polypeptideof the invention having aforementioned the fine chemical increasingactivity to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation.

The hybridization can be by conventional nucleotide complementarity toform a stable duplex, or, for example, in the case of an antisensenucleic acid molecule which binds to DNA duplexes, through specificinteractions in the major groove of the double helix. The antisensenucleic acid molecule can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector in which the antisense nucleic acidmolecule is placed under the control of a strong prokaryotic, viral, oreukaryotic including plant promoters are preferred.

In a further embodiment, the antisense nucleic acid molecule of theinvention can be an α-anomeric nucleic acid molecule. A α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual units, the strands runparallel to each other (Gaultier et al. (1987) Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

Further the antisense nucleic acid molecule of the invention can be alsoa ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity, which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleavemRNA transcripts encoding the polypeptide of the invention to therebyinhibit translation of said mRNA. A ribozyme having specificity for anucleic acid molecule encoding the polypeptide of the invention or usedin the process of the invention can be designed based upon thenucleotide sequence of the nucleic acid molecule of the invention orcoding a protein used in the process of the invention or on the basis ofa heterologous sequence to be isolated according to methods taught inthis invention. For example, a derivative of a Tetrahymena L-19 IVS RNAcan be constructed in which the nucleotide sequence of the active siteis complementary to the nucleotide sequence to be cleaved in an encodingmRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 and Cech et al.U.S. Pat. No. 5,116,742. Alternatively, mRNA encoding the polypeptide ofthe invention or a polypeptide used in the process of the invention canbe used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules. See, e.g., Bartel, D. andSzostak, J. W. (1993) Science 261:1411-1418.

The antisense molecule of the present invention comprises also a nucleicacid molecule comprising a nucleotide sequences complementary to theregulatory region of an nucleotide sequence encoding the naturaloccurring polypeptide of the invention, e.g. the polypeptide sequencesshown in the sequence listing, or identified according to the methodsdescribed herein, e.g., its promoter and/or enhancers, e.g. to formtriple helical structures that prevent transcription of the gene intarget cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6): 569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioassays 14(12):807-15.

Furthermore the present invention relates to a double stranded RNAmolecule capable for the reduction or inhibition of the activity of thegene product of a gene encoding the polypeptide of the invention, apolypeptide used in the process of the invention, the nucleic acidmolecule of the invention or a nucleic acid molecule used in the processof the invention encoding.

The method of regulating genes by means of double-stranded RNA(“double-stranded RNA interference”; dsRNAi) has been describedextensively for animal, yeast, fungi and plant organisms such asNeurospora, zebrafish, Drosophila, mice, planaria, humans, Trypanosoma,petunia or Arabidopsis (for example Matzke M A et al. (2000) Plant Mol.Biol. 43: 401-415; Fire A. et al. (1998) Nature 391: 806-811; WO99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO00/49035; WO 00/63364). In addition RNAi is also documented as anadvantageously tool for the repression of genes in bacteria such as E.coli for example by Tchurikov et al. [J. Biol. Chem., 2000, 275 (34):26523-26529]. Fire et al. named the phenomenon RNAi for “RNAinterference”. The techniques and methods described in the abovereferences are expressly referred to. Efficient gene suppression canalso be observed in the case of transient expression or followingtransient transformation, for example as the consequence of a biolistictransformation (Schweizer P et al. (2000) Plant J 2000 24: 895-903).dsRNAi methods are based on the phenomenon that the simultaneousintroduction of complementary strand and counterstrand of a genetranscript brings about highly effective suppression of the expressionof the gene in question. The resulting phenotype is very similar to thatof an analogous knock-out mutant (Waterhouse P M et al. (1998) Proc.Natl. Acad. Sci. USA 95: 13959-64).

Tuschl et al. [Gens Dev., 1999, 13 (24): 3191-3197] was able to showthat the efficiency of the RNAi method is a function of the length ofthe duplex, the length of the 3′-end overhangs, and the sequence inthese overhangs. Based on the work of Tuschl et al. the followingguidelines can be given to the skilled worker: To achieve good resultsthe 5′ and 3′ untranslated regions of the used nucleic acid sequence andregions close to the start codon should be avoided as this regions arericher in regulatory protein binding sites and interactions between RNAisequences and such regulatory proteins might lead to undesiredinteractions. Preferably a region of the used mRNA is selected, which is50 to 100 nt (=nucleotides or bases) downstream of the AUG start codon.Only dsRNA (=double-stranded RNA) sequences from exons are useful forthe method, as sequences from introns have no effect. The G/C content inthis region should be greater than 30% and less than 70% ideally around50%. A possible secondary structure of the target mRNA is less importantfor the effect of the RNAi method.

The dsRNAi method has proved to be particularly effective andadvantageous for reducing the expression of the nucleic acid sequencesof the SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303,305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331,333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359,361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387,389, 391 or 393 and/or homologs thereof. As described inter alia in WO99/32619, dsRNAi approaches are clearly superior to traditionalantisense approaches. The invention therefore furthermore relates todouble-stranded RNA molecules (dsRNA molecules) which, when introducedinto an organism, advantageously into a plant (or a cell, tissue, organor seed derived therefrom), bring about altered metabolic activity bythe reduction in the expression of the nucleic acid sequences of the SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249,251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277,279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361,363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389,391 or 393 and/or homologs thereof. In a double-stranded RNA moleculefor reducing the expression of an protein encoded by a nucleic acidsequence of one of the SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269,271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325,327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353,355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,383, 385, 387, 389, 391 or 393 and/or homologs thereof, one of the twoRNA strands is essentially identical to at least part of a nucleic acidsequence, and the respective other RNA strand is essentially identicalto at least part of the complementary strand of a nucleic acid sequence.

The term “essentially identical” refers to the fact that the dsRNAsequence may also include insertions, deletions and individual pointmutations in comparison to the target sequence while still bringingabout an effective reduction in expression. Preferably, the homology asdefined above amounts to at least 30%, preferably at least 40%, 50%,60%, 70% or 80%, very especially preferably at least 90%, mostpreferably 100%, between the “sense” strand of an inhibitory dsRNA and apart-segment of a nucleic acid sequence of the invention (or between the“antisense” strand and the complementary strand of a nucleic acidsequence, respectively). The part-segment amounts to at least 10 bases,preferably at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 bases, especially preferably at least 40, 50, 60, 70, 80 or 90bases, very especially preferably at least 100, 200, 300 or 400 bases,most preferably at least 500, 600, 700, 800, 900 or more bases or atleast 1000 or 2000 bases or more in length. In another preferredembodiment of the invention the part-segment amounts to 17, 18, 19, 20,21, 22, 23, 24, 25, 26 or 27 bases, preferably to 20, 21, 22, 23, 24 or25 bases. These short sequences are preferred in animals and plants. Thelonger sequences preferably between 200 and 800 bases are preferred innonmammalian animals, preferably in invertebrates, in yeast, fungi orbacteria, but they are also useable in plants. Long double-stranded RNAsare processed in the organisms into many siRNAs (=small/shortinterfering RNAs) for example by the protein Dicer, which is ads-specific Rnase III enzyme. As an alternative, an “essentiallyidentical” dsRNA may also be defined as a nucleic acid sequence, whichis capable of hybridizing with part of a gene transcript (for example in400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50° C. or 70° C. for 12 to16 h).

The dsRNA may consist of one or more strands of polymerizedribonucleotides. Modification of both the sugar-phosphate backbone andof the nucleosides may furthermore be present. For example, thephosphodiester bonds of the natural RNA can be modified in such a waythat they encompass at least one nitrogen or sulfur heteroatom. Basesmay undergo modification in such a way that the activity of, forexample, adenosine deaminase is restricted. These and othermodifications are described herein below in the methods for stabilizingantisense RNA.

The dsRNA can be prepared enzymatically; it may also be synthesizedchemically, either in full or in part.

The double-stranded structure can be formed starting from a single,self-complementary strand or starting from two complementary strands. Ina single, self-complementary strand, “sense” and “antisense” sequencecan be linked by a linking sequence (“linker”) and form for example ahairpin structure. Preferably, the linking sequence may take the form ofan intron, which is spliced out following dsRNA synthesis. The nucleicacid sequence encoding a dsRNA may contain further elements such as, forexample, transcription termination signals or polyadenylation signals.If the two strands of the dsRNA are to be combined in a cell or anorganism advantageously in a plant, this can be brought about in avariety of ways.

Formation of the RNA duplex can be initiated either outside the cell orwithin the cell. As shown in WO 99/53050, the dsRNA may also encompass ahairpin structure, by linking the “sense” and “antisense” strands by a“linker” (for example an intron). The self-complementary dsRNAstructures are preferred since they merely require the expression of aconstruct and always encompass the complementary strands in an equimolarratio.

The expression cassettes encoding the “antisense” or the “sense” strandof the dsRNA or the self-complementary strand of the dsRNA arepreferably inserted into a vector and stably inserted into the genome ofa plant, using the methods described herein below (for example usingselection markers), in order to ensure permanent expression of thedsRNA.

The dsRNA can be introduced using an amount which makes possible atleast one copy per cell. A larger amount (for example at least 5, 10,100, 500 or 1000 copies per cell) may bring about more efficientreduction.

As has already been described, 100% sequence identity between the dsRNAand a gene transcript of a nucleic acid sequence of one of the SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279,281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363,365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or393 or it's homolog is not necessarily required in order to bring abouteffective reduction in the expression. The advantage is, accordingly,that the method is tolerant with regard to sequence deviations as may bepresent as a consequence of genetic mutations, polymorphisms orevolutionary divergences. Thus, for example, using the dsRNA, which hasbeen generated starting from a sequence of one of SEQ ID NO: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393 or homologsthereof of the one organism, may be used to suppress the correspondingexpression in another organism.

Due to the high degree of sequence homology between sequences fromvarious organisms (e.g. plants), allows the conclusion that theseproteins may be conserved to a high degree within, for example other,plants, it is optionally possible that the expression of a dsRNA derivedfrom one of the disclosed sequences as shown herein or homologs thereofshould also have has an advantageous effect in other plant species.Preferably the consensus sequences shown herein can be used for theconstruction of useful dsRNA molecules.

The dsRNA can be synthesized either in vivo or in vitro. To this end, aDNA sequence encoding a dsRNA can be introduced into an expressioncassette under the control of at least one genetic control element (suchas, for example, promoter, enhancer, silencer, splice donor or spliceacceptor or polyadenylation signal). Suitable advantageous constructsare described herein below. Polyadenylation is not required, nor doelements for initiating translation have to be present.

A dsRNA can be synthesized chemically or enzymatically. Cellular RNApolymerases or bacteriophage RNA polymerases (such as, for example T3,T7 or SP6 RNA polymerase) can be used for this purpose. Suitable methodsfor the in-vitro expression of RNA are described (WO 97/32016; U.S. Pat.No. 5,593,874; U.S. Pat. No. 5,698,425, U.S. Pat. No. 5,712,135, U.S.Pat. No. 5,789,214, U.S. Pat. No. 5,804,693). Prior to introduction intoa cell, tissue or organism, a dsRNA which has been synthesized in vitroeither chemically or enzymatically can be isolated to a higher or lesserdegree from the reaction mixture, for example by extraction,precipitation, electrophoresis, chromatography or combinations of thesemethods. The dsRNA can be introduced directly into the cell or else beapplied extracellularly (for example into the interstitial space).

Advantageously the RNAi method leads to only a partial loss of genefunction and therefore enables the skilled worker to study a gene doseeffect in the disered organism and to fine tune the process of theinvention. Futhermore it enables a person skilled in the art to studymultiple functions of a gene.

Stable transformation of the plant with an expression construct, whichbrings about the expression of the dsRNA is preferred, however. Suitablemethods are described herein below.

A further embodiment of the invention also relates to a method for thegeneration of a transgenic host or host cell, e.g. a eukaryotic orprokaryotic cell, preferably a transgenic microorganism, a transgenicplant cell or a transgenic plant tissue or a transgenic plant, whichcomprises introducing, into the plant, the plant cell or the planttissue, the nucleic acid construct according to the invention, thevector according to the invention, or the nucleic acid moleculeaccording to the invention.

A further embodiment of the invention also relates to a method for thetransient generation of a host or host cell, eukaryotic or prokaryoticcell, preferably a transgenic microorganism, a transgenic plant cell ora transgenic plant tissue or a transgenic plant, which comprisesintroducing, into the plant, the plant cell or the plant tissue, thenucleic acid construct according to the invention, the vector accordingto the invention, the nucleic acid molecule characterized herein asbeing contained in the nucleic acid construct of the invention or thenucleic acid molecule according to the invention, whereby the introducednucleic acid molecules, nucleic acid construct and/or vector is notintegrated into the genome of the host or host cell. Therefore thetransformants are not stable during the propagation of the host inrespect of the introduced nucleic acid molecules, nucleic acid constructand/or vector.

In the process according to the invention, transgenic organisms are alsoto be understood as meaning—if they take the form of plants—plant cells,plant tissues, plant organs such as root, shoot, stem, seed, flower,tuber or leaf, or intact plants which are grown for the production ofthe fine chemical.

Growing is to be understood as meaning for example culturing thetransgenic plant cells, plant tissue or plant organs on or in a nutrientmedium or the intact plant on or in a substrate, for example inhydroponic culture, potting compost or on a field soil.

In a further advantageous embodiment of the process, the nucleic acidmolecules can be expressed in single-celled plant cells (such as algae),see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 andreferences cited therein, and plant cells from higher plants (forexample spermatophytes such as crops). Examples of plant expressionvectors encompass those which are described in detail herein or in:Becker, D. [(1992) Plant Mol. Biol. 20:1195-1197] and Bevan, M. W.[(1984), Nucl. Acids Res. 12:8711-8721; Vectors for Gene Transfer inHigher Plants; in: Transgenic Plants, Vol. 1, Engineering andUtilization, Ed.: Kung and R. Wu, Academic Press, 1993, pp. 15-38]. Anoverview of binary vectors and their use is also found in Hellens, R.[(2000), Trends in Plant Science, Vol. 5 No. 10, 446-451.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. The terms“transformation” and “transfection” include conjugation and transductionand, as used in the present context, are intended to encompass amultiplicity of prior-art methods for introducing foreign nucleic acidmolecules (for example DNA) into a host cell, including calciumphosphate coprecipitation or calcium chloride coprecipitation,DEAE-dextran-mediated transfection, PEG-mediated transfection,lipofection, natural competence, chemically mediated transfer,electroporation or particle bombardment. Suitable methods for thetransformation or transfection of host cells, including plant cells, canbe found in Sambrook et al. (Molecular Cloning: A Laboratory Manual.,2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) and in other laboratory handbookssuch as Methods in Molecular Biology, 1995, Vol. 44, Agrobacteriumprotocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.

The above-described methods for the transformation and regeneration ofplants from plant tissues or plant cells are exploited for transient orstable transformation of plants. Suitable methods are the transformationof protoplasts by polyethylene-glycol-induced DNA uptake, the biolisticmethod with the gene gun—known as the particle bombardment method—,electroporation, the incubation of dry embryos in DNA-containingsolution, microinjection and the Agrobacterium-mediated gene transfer.The abovementioned methods are described for example in B. Jenes,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, edited by S. D. Kung and R. Wu, Academic Press (1993)128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991) 205-225. The construct to be expressed is preferably cloned intoa vector, which is suitable for transforming Agrobacterium tumefaciens,for example pBin19 (Bevan, Nucl. Acids Res. 12 (1984) 8711).Agrobacteria transformed with such a vector can then be used in theknown manner for the transformation of plants, in particular cropplants, such as, for example, tobacco plants, for example by bathingscarified leaves or leaf segments in an agrobacterial solution andsubsequently culturing them in suitable media. The transformation ofplants with Agrobacterium tumefaciens is described for example by Höfgenand Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or known from, interalia, F. F. White, Vectors for Gene Transfer in Higher Plants; inTransgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D.Kung and R. Wu, Academic Press, 1993, pp. 15-38.

To select for the successful transfer of the nucleic acid molecule,vector or nucleic acid construct of the invention according to theinvention into a host organism, it is advantageous to use marker genesas have already been described above in detail. It is known of thestable or transient integration of nucleic acids into plant cells thatonly a minority of the cells takes up the foreign DNA and, if desired,integrates it into its genome, depending on the expression vector usedand the transfection technique used. To identify and select theseintegrants, a gene encoding for a selectable marker (as described above,for example resistance to antibiotics) is usually introduced into thehost cells together with the gene of interest. Preferred selectablemarkers in plants comprise those, which confer resistance to anherbicide such as glyphosate or gluphosinate. Other suitable markersare, for example, markers, which encode genes involved in biosyntheticpathways of, for example, sugars or amino acids, such asβ-galactosidase, ura3 or ilv2. Markers, which encode genes such asluciferase, gfp or other fluorescence genes, are likewise suitable.These markers and the aforementioned markers can be used in mutants inwhom these genes are not functional since, for example, they have beendeleted by conventional methods. Furthermore, nucleic acid molecules,which encode a selectable marker, can be introduced into a host cell onthe same vector as those, which encode the polypeptides of the inventionor used in the process or else in a separate vector. Cells which havebeen transfected stably with the nucleic acid introduced can beidentified for example by selection (for example, cells which haveintegrated the selectable marker survive whereas the other cells die).

Since the marker genes, as a rule specifically the gene for resistanceto antibiotics and herbicides, are no longer required or are undesiredin the transgenic host cell once the nucleic acids have been introducedsuccessfully, the process according to the invention for introducing thenucleic acids advantageously employs techniques which enable theremoval, or excision, of these marker genes. One such a method is whatis known as cotransformation. The cotransformation method employs twovectors simultaneously for the transformation, one vector bearing thenucleic acid according to the invention and a second bearing the markergene(s). A large proportion of transformants receives or, in the case ofplants, comprises (up to 40% of the transformants and above), bothvectors. In case of transformation with Agrobacteria, the transformantsusually receive only a part of the vector, the sequence flanked by theT-DNA which usually represents the expression cassette. The marker genescan subsequently be removed from the transformed plant by performingcrosses. In another method, marker genes integrated into a transposonare used for the transformation together with desired nucleic acid(known as the Ac/Ds technology). The transformants can be crossed with atransposase resource or the transformants are transformed with a nucleicacid construct conferring expression of a transposase, transiently orstable. In some cases (approx. 10%), the transposon jumps out of thegenome of the host cell once transformation has taken place successfullyand is lost. In a further number of cases, the transposon jumps to adifferent location. In these cases, the marker gene must be eliminatedby performing crosses. In microbiology, techniques were developed whichmake possible, or facilitate, the detection of such events. A furtheradvantageous method relies on what are known as recombination systems,whose advantage is that elimination by crossing can be dispensed with.The best-known system of this type is what is known as the Cre/loxsystem. Cre1 is a recombinase, which removes the sequences locatedbetween the loxP sequences. If the marker gene is integrated between theloxP sequences, it is removed, once transformation has taken placesuccessfully, by expression of the recombinase. Further recombinationsystems are the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J.Biol. Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol.,149, 2000: 553-566). A site-specific integration into the plant genomeof the nucleic acid sequences according to the invention is possible.Naturally, these methods can also be applied to microorganisms such asyeast, fungi or bacteria.

Agrobacteria transformed with an expression vector according to theinvention may also be used in the manner known per se for thetransformation of plants such as experimental plants like Arabidopsis orcrop plants, such as, for example, cereals, maize, oats, rye, barley,wheat, soya, rice, cotton, sugarbeet, canola, sunflower, flax, hemp,potato, tobacco, tomato, carrot, bell peppers, oilseed rape, tapioca,cassava, arrow root, tagetes, alfalfa, lettuce and the various tree,nut, and grapevine species, in particular oil-containing crop plantssuch as soya, peanut, castor-oil plant, sunflower, maize, cotton, flax,oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius) orcocoa beans, for example by bathing scarified leaves or leaf segments inan agrobacterial solution and subsequently growing them in suitablemedia.

In addition to the transformation of somatic cells, which then has to beregenerated into intact plants, it is also possible to transform thecells of plant meristems and in particular those cells which developinto gametes. In this case, the transformed gametes follow the naturalplant development, giving rise to transgenic plants. Thus, for example,seeds of Arabidopsis are treated with agrobacteria and seeds areobtained from the developing plants of which a certain proportion istransformed and thus transgenic (Feldman, K A and Marks M D (1987). MolGen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J.Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore,pp. 274-289). Alternative methods are based on the repeated removal ofthe influorescences and incubation of the excision site in the center ofthe rosette with transformed agrobacteria, whereby transformed seeds canlikewise be obtained at a later point in time (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, anespecially effective method is the vacuum infiltration method with itsmodifications such as the “floral dip” method. In the case of vacuuminfiltration of Arabidopsis, intact plants under reduced pressure aretreated with an agrobacterial suspension (Bechthold, N (1993). C R AcadSci Paris Life Sci, 316: 1194-1199), while in the case of the “floraldip” method the developing floral tissue is incubated briefly with asurfactant-treated agrobacterial suspension (Clough, S J und Bent, A F(1998). The Plant J. 16, 735-743). A certain proportion of transgenicseeds are harvested in both cases, and these seeds can be distinguishedfrom nontransgenic seeds by growing under the above-described selectiveconditions. In addition the stable transformation of plastids is ofadvantages because plastids are inherited maternally is most cropsreducing or eliminating the risk of transgene flow through pollen. Thetransformation of the chloroplast genome is generally achieved by aprocess, which has been schematically displayed in Klaus et al., 2004(Nature Biotechnology 22(2), 225-229). Briefly the sequences to betransformed are cloned together with a selectable marker gene betweenflanking sequences homologous to the chloroplast genome. Thesehomologous flanking sequences direct site-specific integration into theplastome. Plastidal transformation has been described for many differentplant species and an overview can be taken from Bock (2001) Transgenicplastids in basic research and plant biotechnology. J Mol Biol. 2001Sep. 21; 312(3): 425-38 or Maliga, P (2003) Progress towardscommercialization of plastid transformation technology. TrendsBiotechnol. 21, 20-28. Further biotechnological progress has recentlybeen reported in form of marker free plastid transformants, which can beproduced by a transient cointegrated maker gene (Klaus et al., 2004,Nature Biotechnology 22(2), 225-229).

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the abovementioned publications of S. D. Kung and R. Wu, Potrykus orHöfgen and Willmitzer.

Accordingly, the present invention thus also relates to a plant cellcomprising the nucleic acid construct according to the invention, thenucleic acid molecule according to the invention or the vector accordingto the invention.

Accordingly the present invention relates to any cell transgenic for anynucleic acid characterized as part of the invention, e.g. conferring theincrease of the fine chemical in a cell or an organism or a partthereof, e.g. the nucleic acid molecule of the invention, the nucleicacid construct of the invention, the antisense molecule of theinvention, the vector of the invention or a nucleic acid moleculeencoding the polypeptide of the invention, e.g. encoding a polypeptidehaving the biological activity of the protein of the invention. Due tothe above mentioned activity the fine chemical content in a cell or anorganism is increased. For example, due to modulation or manupulation,the cellular activity is increased, e.g. due to an increased expressionor specific activity of the subject matters of the invention in a cellor an organism or a part thereof. Transgenic for a polypeptide havingbiological activity of the protein of the invention or activity meansherein that due to modulation or manipulation of the genome, thebiological activity of the protein of the invention is increased in thecell or organism or part thereof. Examples are described above incontext with the process of the invention

“Transgenic”, for example regarding a nucleic acid molecule, an nucleicacid construct or a vector comprising said nucleic acid molecule or anorganism transformed with said nucleic acid molecule, nucleic acidconstruct or vector, refers to all those subjects originating byrecombinant methods in which either

-   a) the nucleic acid sequence, or-   b) a genetic control sequence linked operably to the nucleic acid    sequence, for example a promoter, or-   c) (a) and (b)    are not located in their natural genetic environment or have been    modified by recombinant methods, an example of a modification being    a substitution, addition, deletion, inversion or insertion of one or    more nucleotide residues. Natural genetic environment refers to the    natural chromosomal locus in the organism of origin, or to the    presence in a genomic library. In the case of a genomic library, the    natural genetic environment of the nucleic acid sequence is    preferably retained, at least in part. The environment flanks the    nucleic acid sequence at least at one side and has a sequence of at    least 50 bp, preferably at least 500 bp, especially preferably at    least 1000 bp, very especially preferably at least 5000 bp, in    length.

A naturally occurring expression cassette—for example the naturallyoccurring combination of the promoter of the protein of the inventionwith the corresponding protein gene—becomes a transgenic expressioncassette when it is modified by non-natural, synthetic “artificial”methods such as, for example, mutagenization. Such methods have beendescribed (U.S. Pat. No. 5,565,350; WO 00/15815; also see above).

Further, the plant cell, plant tissue or plant can also be transformedsuch that further enzymes and proteins are (over)expressed whichexpression supports an increase of the fine chemical.

However, transgenic also means that the nucleic acids according to theinvention are located at their natural position in the genome of anorganism, but that the sequence has been modified in comparison with thenatural sequence and/or that the regulatory sequences of the naturalsequences have been modified. Preferably, transgenic/recombinant is tobe understood as meaning the transcription of the nucleic acids used inthe process according to the invention occurs at a non-natural positionin the genome, that is to say the expression of the nucleic acids ishomologous or, preferably, heterologous. This expression can betransiently or of a sequence integrated stably into the genome.

The term “transgenic plants” used in accordance with the invention alsorefers to the progeny of a transgenic plant, for example the T₁, T₂, T₃and subsequent plant generations or the BC₁, BC₂, BC₃ and subsequentplant generations. Thus, the transgenic plants according to theinvention can be raised and selfed or crossed with other individuals inorder to obtain further transgenic plants according to the invention.Transgenic plants may also be obtained by propagating transgenic plantcells vegetatively. The present invention also relates to transgenicplant material, which can be derived from a transgenic plant populationaccording to the invention. Such material includes plant cells andcertain tissues, organs and parts of plants in all their manifestations,such as seeds, leaves, anthers, fibers, tubers, roots, root hairs,stems, embryo, calli, cotelydons, petioles, harvested material, planttissue, reproductive tissue and cell cultures, which are derived fromthe actual transgenic plant and/or can be used for bringing about thetransgenic plant.

Any transformed plant obtained according to the invention can be used ina conventional breeding scheme or in in vitro plant propagation toproduce more transformed plants with the same characteristics and/or canbe used to introduce the same characteristic in other varieties of thesame or related species. Such plants are also part of the invention.Seeds obtained from the transformed plants genetically also contain thesame characteristic and are part of the invention. As mentioned before,the present invention is in principle applicable to any plant and cropthat can be transformed with any of the transformation method known tothose skilled in the art. Another embodiment of the invention is the useof the nucleic acid molecule as claimed in above in mapping and breedingprocesses for the identification of plant varieties having and increasedcapacity for production of the fine chemical.

In an especially preferred embodiment, the organism, the host cell,plant cell, plant, microorganism or plant tissue according to theinvention is transgenic.

Accordingly, the invention therefore relates to transgenic organismstransformed with at least one nucleic acid molecule, nucleic acidconstruct or vector according to the invention, and to cells, cellcultures, tissues, parts—such as, for example, in the case of plantorganisms, plant tissue, for example leaves, roots and the like—orpropagation material derived from such organisms, or intact plants. Theterms “recombinant (host)”, and “transgenic (host)” are usedinterchangeably in this context. Naturally, these terms refer not onlyto the host organism or target cell in question, but also to theprogeny, or potential progeny, of these organisms or cells. Sincecertain modifications may occur in subsequent generations owing tomutation or environmental effects, such progeny is not necessarilyidentical with the parental cell, but still comes within the scope ofthe term as used herein.

Suitable organisms for the process according to the invention or ashosts are all these eukaryotic or prokaryotic organisms, which arecapable of synthesizing the fine chemcial. The organisms used as hostsare microorganisms, such as bacteria, fungi, yeasts or algae, non-humananimals, or plants, such as dictotyledonous or monocotyledonous plants.

In principle all plants can be used as host organism, especially theplants mentioned above as source organism. Preferred transgenic plantsare, for example, selected from the families Aceraceae, Anacardiaceae,Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae,Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae, Papaveraceae,Rosaceae, Salicaceae, Solanaceae, Arecaceae, Bromeliaceae, Cyperaceae,Iridaceae, Liliaceae, Orchidaceae, Gentianaceae, Labiaceae,Magnoliaceae, Ranunculaceae, Carifolaceae, Rubiaceae, Scrophulariaceae,Caryophyllaceae, Ericaceae, Polygonaceae, Violaceae, Juncaceae orPoaceae and preferably from a plant selected from the group of thefamilies Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae,Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae. Preferred arecrop plants such as plants advantageously selected from the group of thegenus peanut, oilseed rape, canola, sunflower, safflower, olive, sesame,hazelnut, almond, avocado, bay, pumpkin/squash, linseed, soya,pistachio, borage, maize, wheat, rye, oats, sorghum and millet,triticale, rice, barley, cassava, potato, sugarbeet, egg plant, alfalfa,and perennial grasses and forage plants, oil palm, vegetables(brassicas, root vegetables, tuber vegetables, pod vegetables, fruitingvegetables, onion vegetables, leafy vegetables and stem vegetables),buckwheat, Jerusalem artichoke, broad bean, vetches, lentil, dwarf bean,lupin, clover and Lucerne for mentioning only some of them.

Preferred plant cells, plant organs, plant tissues or parts of plantsoriginate from the under source organism mentioned plant families,preferably from the abovementioned plant genus, more preferred fromabovementioned plants spezies.

Transgenic plants comprising the fine chemical synthesized in theprocess according to the invention can be marketed directly withoutisolation of the compounds synthesized. In the process according to theinvention, plants are understood as meaning all plant parts, plantorgans such as leaf, stalk, root, tubers or seeds or propagationmaterial or harvested material or the intact plant. In this context, theseed encompasses all parts of the seed such as the seed coats, epidermalcells, seed cells, endosperm or embryonic tissue. The fine chemicalproduced in the process according to the invention may, however, also beisolated from the plant in free or bound form. The fine chemicalproduced by this process can be harvested by harvesting the organismseither from the culture in which they grow or from the field. This canbe done via expressing, grinding and/or extraction, salt precipitationand/or ion-exchange chromatography of the plant parts, preferably theplant seeds, plant fruits, plant tubers and the like.

In a further embodiment, the present invention relates to a process forthe generation of a microorganism, comprising the introduction, into themicroorganism or parts thereof, of the nucleic acid construct of theinvention, or the vector of the invention or the nucleic acid moleculeof the invention.

In another embodiment, the present invention relates also to atransgenic microorganism comprising the nucleic acid molecule of theinvention, the nucleic acid construct of the invention or the vector asof the invention. Appropriate microorganisms have been described hereinbefore under source organism, preferred are in particular aforementionedstrains suitable for the production of fine chemicals.

Accordingly, the present invention relates also to a process accordingto the present invention whereby the produced fine chemical or finechemical composition is isolated.

In this manner, more than 50% by weight, advantageously more than 60% byweight, preferably more than 70% by weight, especially preferably morethan 80% by weight, very especially preferably more than 90% by weight,of the fine chemical produced in the process can be isolated. Theresulting fine chemical can, if appropriate, subsequently be furtherpurified, if desired mixed with other active ingredients such asvitamins, amino acids, carbohydrates, antibiotics and the like, and, ifappropriate, formulated.

The fine chemical obtained in the process is suitable as startingmaterial for the synthesis of further products of value. For example,they can be used in combination with other ingredients or alone for theproduction of pharmaceuticals, foodstuffs, animal feeds or cosmetics.Accordingly, the present invention relates a method for the productionof a pharmaceuticals, food stuff, animal feeds, nutrients or cosmeticscomprising the steps of the process according to the invention,including the isolation of the fine chemical or fine chemicalcomposition produced and if desired formulating the product with apharmaceutical acceptable carrier or formulating the product in a formacceptable for an application in agriculture. A further embodimentaccording to the invention is the use of the fine chemical produced inthe process or of the transgenic organisms in animal feeds, foodstuffs,medicines, food supplements, cosmetics or pharmaceuticals.

In principle all microorganisms can be used as host organism especiallythe ones mentioned under source organism above. It is advantageous touse in the process of the invention transgenic microorganisms such asfungi such as the genus Claviceps or Aspergillus or Gram-positivebacteria such as the genera Bacillus, Corynebacterium, Micrococcus,Brevibacterium, Rhodococcus, Nocardia, Caseobacter or Arthrobacter orGram-negative bacteria such as the genera Escherichia, Flavobacterium orSalmonella or yeasts such as the genera Rhodotorula, Saccharomyces,Hansenula or Candida. Particularly advantageous organisms are selectedfrom the group of genera Corynebacterium, Brevibacterium, Escherichia,Bacillus, Rhodotorula, Saccharomyces, Hansenula, Candida, Claviceps orFlavobacterium. It is very particularly advantageous to use in theprocess of the invention microorganisms selected from the group ofgenera and species consisting of Saccharomyces cerevisiae, Hansenulaanomala, Candida utilis, Claviceps purpurea, Bacillus circulans,Bacillus subtilis, Bacillus sp., Brevibacterium albidum, Brevibacteriumalbum, Brevibacterium cerinum, Brevibacterium flavum, Brevibacteriumglutamigenes, Brevibacterium iodinum, Brevibacterium ketoglutamicum,Brevibacterium lactofermentum, Brevibacterium linens, Brevibacteriumroseum, Brevibacterium saccharolyticum, Brevibacterium sp.,Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum,Corynebacterium ammoniagenes, Corynebacterium glutamicum (=Micrococcusglutamicum), Corynebacterium melassecola, Corynebacterium sp. orEscherichia coli, specifically Saccharomyces cerevisiae, Escherichiacoli K12 and its described strains.

The process of the invention is, when the host organisms aremicroorganisms, advantageously carried out at a temperature between 0°C. and 95° C., preferably between 10° C. and 85° C., particularlypreferably between 15° C. and 75° C., very particularly preferablybetween 15° C. and 45° C. The pH is advantageously kept at between pH 4and 12, preferably between pH 6 and 9, particularly preferably betweenpH 7 and 8, during this. The process of the invention can be operatedbatchwise, semibatchwise or continuously. A summary of known cultivationmethods is to be found in the textbook by Chmiel (Bioprozeβtechnik 1.Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag,Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren undperiphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).The culture medium to be used must meet the requirements of therespective strains in a suitable manner. Descriptions of culture mediafor various microorganisms are present in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981). These media, which can beemployed according to the invention include, as described above, usuallyone or more carbon sources, nitrogen sources, inorganic salts, vitaminsand/or trace elements. Preferred carbon sources are sugars such asmono-, di- or polysaccharides. Examples of very good carbon sources areglucose, fructose, mannose, galactose, ribose, sorbose, ribulose,lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars canalso be added to the media via complex compounds such as molasses, orother byproducts of sugar refining. It may also be advantageous to addmixtures of various carbon sources. Other possible carbon sources areoils and fats such as, for example, soybean oil, sunflower oil, peanutoil and/or coconut fat, fatty acids such as, for example, palmitic acid,stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as,for example, glycerol, methanol and/or ethanol and/or organic acids suchas, for example, acetic acid and/or lactic acid. Nitrogen sources areusually organic or inorganic nitrogen compounds or materials, whichcontain these compounds. Examples of nitrogen sources include ammonia inliquid or gaseous form or ammonium salts such as ammonium sulfate,ammonium chloride, ammonium phosphate, ammonium carbonate or ammoniumnitrate, nitrates, urea, amino acids or complex nitrogen sources such ascorn steep liquor, soybean meal, soybean protein, yeast extract, meatextract and others. The nitrogen sources may be used singly or as amixture. Inorganic salt compounds, which may be present in the mediainclude the chloride, phosphorus or sulfate salts of calcium, magnesium,sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

For preparing sulfur-containing fine chemicals, in particular the finechemical, it is possible to use as sulfur source inorganicsulfur-containing compounds such as, for example, sulfates, sulfites,dithionites, tetrathionates, thiosulfates, sulfides or else organicsulfur compounds such as mercaptans and thiols.

It is possible to use as phosphorus source phosphoric acid, potassiumdihydrogenphosphate or dipotassium hydrogenphosphate or thecorresponding sodium-containing salts. Chelating agents can be added tothe medium in order to keep the metal ions in solution. Particularlysuitable chelating agents include dihydroxyphenols such as catechol orprotocatechuate, or organic acids such as citric acid. The fermentationmedia employed according to the invention for cultivating microorganismsnormally also contain other growth factors such as vitamins or growthpromoters, which include, for example, biotin, riboflavin, thiamine,folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factorsand salts are often derived from complex media components such as yeastextract, molasses, corn steep liquor and the like. Suitable precursorscan moreover be added to the culture medium. The exact composition ofthe media compounds depends greatly on the particular experiment and ischosen individually for each specific case. Information about mediaoptimization is obtainable from the textbook “Applied Microbiol.Physiology, A Practical Approach” (editors P. M. Rhodes, P. F. Stanbury,IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can alsobe purchased from commercial suppliers such as Standard 1 (Merck) or BHI(Brain heart infusion, DIFCO) and the like. All media components aresterilized either by heat (1.5 bar and 121° C. for 20 min) or bysterilizing filtration. The components can be sterilized either togetheror, if necessary, separately. All media components can be present at thestart of the cultivation or optionally be added continuously orbatchwise. The temperature of the culture is normally between 15° C. and45° C., preferably at 25° C. to 40° C., and can be kept constant orchanged during the experiment. The pH of the medium should be in therange from 5 to 8.5, preferably around 7. The pH for the cultivation canbe controlled during the cultivation by adding basic compounds such assodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia oracidic compounds such as phosphoric acid or sulfuric acid. Foaming canbe controlled by employing antifoams such as, for example fatty acidpolyglycol esters. The stability of plasmids can be maintained by addingto the medium suitable substances having a selective effect, for exampleantibiotics. Aerobic conditions are maintained by introducing oxygen oroxygen-containing gas mixtures such as, for example ambient air into theculture. The temperature of the culture is normally from 20° C. to 45°C. and preferably from 25° C. to 40° C. The culture is continued untilformation of the desired product is at a maximum. This aim is normallyachieved within 10 hours to 160 hours.

The fermentation broths obtained in this way, containing the finechemical in particular for example amino acids such as L-methionine,L-threonine and/or L-lysine, normally have a dry matter content of from7.5 to 25% by weight. Sugar-limited fermentation is additionallyadvantageous, at least at the end, but especially over at least 30% ofthe fermentation time. This means that the concentration of utilizablesugar in the fermentation medium is kept at, or reduced to, ≧0 to 3 g/lduring this time. The fermentation broth is then processed further.Depending on requirements, the biomass can be removed entirely or partlyby separation methods, such as, for example, centrifugation, filtration,decantation or a combination of these methods, from the fermentationbroth or left completely in it. The fermentation broth can then bethickened or concentrated by known methods, such as, for example, withthe aid of a rotary evaporator, thin-film evaporator, falling filmevaporator, by reverse osmosis or by nanofiltration. This concentratedfermentation broth can then be worked up by freeze-drying, spray drying,spray granulation or by other processes.

However, it is also possible to purify the fine chemical producedfurther. For this purpose, the product-containing composition issubjected to a chromatography on a suitable resin, in which case thedesired product or the impurities are retained wholly or partly on thechromatography resin. These chromatography steps can be repeated ifnecessary, using the same or different chromatography resins. Theskilled worker is familiar with the choice of suitable chromatographyresins and their most effective use. The purified product can beconcentrated by filtration or ultrafiltration and stored at atemperature at which the stability of the product is a maximum.

The identity and purity of the isolated compound(s) can be determined byprior art techniques. These include high performance liquidchromatography (HPLC), spectroscopic methods, mass spectrometry (MS),staining methods, thin-layer chromatography, NIRS, enzyme assay ormicrobiological assays. These analytical methods are summarized in:Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova etal. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of IndustrialChemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp.540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology,John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC inBiochemistry in: Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 17.

In yet another aspect, the invention also relates to harvestable partsand to propagation material of the transgenic plants according to theinvention which either contain transgenic plant cells expressing anucleic acid molecule according to the invention or which contains cellswhich show an increased cellular activity of the polypeptide of theinvention, e.g. an increased expression level or higher activity of thedescribed protein.

Harvestable parts can be in principle any useful parts of a plant, forexample, flowers, pollen, seedlings, tubers, leaves, stems, fruit,seeds, roots etc. Propagation material includes, for example, seeds,fruits, cuttings, seedlings, tubers, rootstocks etc. Preferred areseeds, fruits, seedlings or tubers as harvestable or propagationmaterial.

The invention furthermore relates to the use of the transgenic organismsaccording to the invention and of the cells, cell cultures, parts—suchas, for example, roots, leaves and the like as mentioned above in thecase of transgenic plant organisms—derived from them, and to transgenicpropagation material such as seeds or fruits and the like as mentionedabove, for the production of foodstuffs or feeding stuffs,pharmaceuticals or fine chemicals.

Accordingly in another embodiment, the present invention relates to theuse of the nucleic acid molecule, the organism, e.g. the microorganism,the plant, plant cell or plant tissue, the vector, or the polypeptide ofthe present invention for making fine chemicals such as fatty acids,carotenoids, isoprenoids, vitamins, lipids, wax esters,(poly)saccharides and/or polyhydroxyalkanoates, and/or its metabolismproducts, in particular, steroid hormones, cholesterol, prostaglandin,triacylglycerols, bile acids and/or ketone bodies producing cells,tissues and/or plants. There are a number of mechanisms by which theyield, production, and/or efficiency of production of fatty acids,carotenoids, isoprenoids, vitamins, wax esters, lipids,(poly)saccharides and/or polyhydroxyalkanoates, and/or its metabolismproducts, in particular, steroid hormones, cholesterol,triacylglycerols, prostaglandin, bile acids and/or ketone bodies orfurther of above defined fine chemicals incorporating such an alteredprotein can be affected. In the case of plants, by e.g. increasing theexpression of acetyl-CoA which is the basis for many products, e.g.,fatty acids, carotenoids, isoprenoids, vitamines, lipids,(poly)saccharides, wax esters, and/or polyhydroxyalkanoates, and/or itsmetabolism products, in particular, prostaglandin, steroid hormones,cholesterol, triacylglycerols, bile acids and/or ketone bodies in acell, it may be possible to increase the amount of the produced saidcompounds thus permitting greater ease of harvesting and purification orin case of plants more efficient partitioning. Further, one or more ofsaid metabolism products, increased amounts of the cofactors, precursormolecules, and intermediate compounds for the appropriate biosyntheticpathways maybe required. Therefore, by increasing the number and/oractivity of transporter proteins involved in the import of nutrients,such as carbon sources (i.e., sugars), nitrogen sources (i.e., aminoacids, ammonium salts), phosphate, and sulfur, it may be possible toimprove the production of acetyl CoA and its metabolism products asmentioned above, due to the removal of any nutrient supply limitationson the biosynthetic process. In particular, it may be possible toincrease the yield, production, and/or efficiency of production of saidcompounds, e.g. fatty acids, carotenoids, isoprenoids, vitamins, wasesters, lipids, (poly)saccharides, and/or polyhydroxyalkanoates, and/orits metabolism products, in particular, steroid hormones, cholesterol,prostaglandin, triacylglycerols, bile acids and/or ketone bodiesmolecules etc. in plants.

Furthermore preferred is a method for the recombinant production ofpharmaceuticals or fine chemicals in host organisms, wherein a hostorganism is transformed with one of the above-described nucleic acidconstructs comprising one or more structural genes which encode thedesired fine chemical or catalyze the biosynthesis of the desired finechemical, the transformed host organism is cultured, and the desiredfine chemical is isolated from the culture medium. This method can beapplied widely to fine chemicals such as enzymes, vitamins, amino acids,sugars, fatty acids, and natural and synthetic flavorings, aromasubstances and colorants or compositions comprising these. Especiallypreferred is the additional production of further amino acids,tocopherols and tocotrienols and carotenoids or compositions comprisingsaid compounds. The transformed host organisms are cultured and theproducts are recovered from the host organisms or the culture medium bymethods known to the skilled worker or the organism itself servers asfood or feed supplement. The production of pharmaceuticals such as, forexample, antibodies or vaccines, is described by Hood E E, Jilka J M.Curr Opin Biotechnol. 1999 Aug.; 10(4): 382-6; Ma J K, Vine N D. CurrTop Microbiol Immunol. 1999; 236:275-92.

In one embodiment, the present invention relates to a method for theidentification of a gene product conferring an increase in the finechemical production in a cell, comprising the following steps:

-   (a) contacting, e.g. hybridising, the nucleic acid molecules of a    sample, e.g. cells, tissues, plants or microorganisms or a nucleic    acid library, which can contain a candidate gene encoding a gene    product conferring an increase in the fine chemical after    expression, with the nucleic acid molecule of the present invention;-   (b) identifying the nucleic acid molecules, which hybridize under    relaxed stringent conditions with the nucleic acid molecule of the    present invention in particular to the nucleic acid molecule    sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,    23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63,    65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,    99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,    127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,    153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,    179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,    205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,    231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,    257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281,    283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,    309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,    335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359,    361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385,    387, 389, 391 or 393 and, optionally, isolating the full length cDNA    clone or complete genomic clone;-   (c) introducing the candidate nucleic acid molecules in host cells,    preferably in a plant cell or a microorganism, appropriate for    producing the fine chemical;-   (d) expressing the identified nucleic acid molecules in the host    cells;-   (e) assaying the the fine chemical level in the host cells; and-   (f) identifying the nucleic acid molecule and its gene product which    expression confers an increase in the the fine chemical level in the    host cell after expression compared to the wild type.

Relaxed hybridisation conditions are: After standard hybridisationprocedures washing steps can be performed at low to medium stringencyconditions usually with washing conditions of 40°-55° C. and saltconditions between 2×SSC and 0.2×SSC with 0.1% SDS in comparison tostringent washing conditions as e.g. 60°-68° C. with 0.1% SDS. Furtherexamples can be found in the references listed above for the stringendhybridization conditions. Usually washing steps are repeated withincreasing stringency and length until a useful signal to noise ratio isdetected and depend on many factors as the target, e.g. its purity,GC-content, size etc, the probe, e.g. its length, is it a RNA or a DNAprobe, salt conditions, washing or hybridisation temperature, washing orhybridisation time etc.

In another embodiment, the present invention relates to a method for theidentification of a gene product conferring an increase in the finechemical production in a cell, comprising the following steps:

-   (a) identifiying nucleic acid molecules of an organism; which can    contain a candidate gene encoding a gene product conferring an    increase in the fine chemical after expression, which are at least    20%, preferably 25%, more preferably 30%, even more preferred are    35%, 40% or 50%, even more preferred are 60%, 70% or 80%, most    preferred are 90% or 95% or more homology to the nucleic acid    molecule of the present invention, for example via homology search    in a data bank;-   (b) introducing the candidate nucleic acid molecules in host cells,    preferably in a plant cells or microorganisms, appropriate for    producing the fine chemical;-   (c) expressing the identified nucleic acid molecules in the host    cells;-   (d) assaying the the fine chemcial level in the host cells; and-   (e) identifying the nucleic acid molecule and its gene product which    expression confers an increase in the the fine chemical level in the    host cell after expression compared to the wild type.

The nucleic acid molecules identified can then be used for theproduction of the fine chemical in the same way as the nucleic acidmolecule of the present invention. Accordingly, in one embodiment, thepresent invention relates to a process for the production of the finechemical, comprising (a) identifying a nucleic acid molecule accordingto aforementioned steps (a) to (f) or (a) to (e) and recovering the freeor bound fine chemical from a organism having an increased cellularactivity of a polypeptide encoded by the isolated nucleic acid moleculecompared to a wild type.

Furthermore, in one embodiment, the present invention relates to amethod for the identification of a compound stimulating production ofthe fine chemical to said plant comprising:

-   a) contacting cells which express the polypeptide of the present    invention or its mRNA with a candidate compound under cell    cultivation conditions;-   b) assaying an increase in expression of said polypeptide or said    mRNA;-   c) comparing the expression level to a standard response made in the    absence of said candidate compound; whereby, an increased expression    over the standard indicates that the compound is stimulating    production of the fine chemical.

Furthermore, in one embodiment, the present invention relates to amethod for the screening for agonists or an antagonist of the activityof the polypeptide of the present invention or used in the process ofthe present invention, e.g. a polypeptide conferring an increase of thefine chemical in an organism or a part therof after increasing theactivity in an organism or a part thereof, comprising:

-   (a) contacting cells, tissues, plants or microorganisms which    express the polypeptide according to the invention with a candidate    compound or a sample comprising a plurality of compounds under    conditions which permit the expression the polypeptide of the    present invention or used in the process of the present invention;-   (b) assaying the fine chemical level or the polypeptide expression    level in the cell, tissue, plant or microorganism or the media the    cell, tissue, plant or microorganisms is cultured or maintained in;    and-   (c) identifying a agonist or antagonist by comparing the measured    the fine chemical level or polypeptide of the invention or used in    the invention expression level with a standard the fine chemical or    polypeptide expression level measured in the absence of said    candidate compound or a sample comprising said plurality of    compounds, whereby an increased level over the standard indicates    that the compound or the sample comprising said plurality of    compounds is an agonist and a decreased level over the standard    indicates that the compound or the sample comprising said plurality    of compounds is an antagonist.

Furthermore, in one embodiment, the present invention relates to processfor the identification of a compound conferring increased the finechemical production in a plant or microorganism, comprising the steps:

-   (a) culturing a cell or tissue or microorganism or maintaining a    plant expressing the polypeptide according to the invention or a    nucleic acid molecule encoding said polypeptide and a readout system    capable of interacting with the polypeptide under suitable    conditions which permit the interaction of the polypeptide with said    readout system in the presence of a compound or a sample comprising    a plurality of compounds and capable of providing a detectable    signal in response to the binding of a compound to said polypeptide    under conditions which permit the expression of said readout system    and the polypeptide of the present invention or used in the process    of the invention; and-   (b) identifying if the compound is an effective agonist by detecting    the presence or absence or increase of a signal produced by said    readout system.

The screen for a gene product or an agonist conferring an increase inthe fine chemical production can be performed by growth of an organismfor example a microorganism in the presence of growth reducing amountsof an inhibitor of the synthesis of the fine chemical. Better growth, eghigher dividing rate or high dry mass in comparison to the control undersuch conditions would identify a gene or gene product or an agonistconferring an increase in fine chemical production.

One can think to screen for increased fine chemical production by forexample resistance to a drug blocking the fine chemical synthesis andlooking whether this effect is dependent on the protein of the inventione.g. comparing near identical organisms with low and high biologicalacitivity of the protein of the invention.

Said compound may be chemically synthesized or microbiologicallyproduced and/or comprised in, for example, samples, e.g., cell extractsfrom, e.g., plants, animals or microorganisms, e.g. pathogens.Furthermore, said compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating the polypeptide of thepresent invention. The reaction mixture may be a cell free extract ormay comprise a cell or tissue culture. Suitable set ups for the methodof the invention are known to the person skilled in the art and are, forexample, generally described in Alberts et al., Molecular Biology of theCell, third edition (1994), in particular Chapter 17. The compounds maybe, e.g., added to the reaction mixture, culture medium, injected intothe cell or sprayed onto the plant.

If a sample containing a compound is identified in the method of theinvention, then it is either possible to isolate the compound from theoriginal sample identified as containing the compound capable ofactivating or increasing the content of the fine chemical in an organismor part thereof, or one can further subdivide the original sample, forexample, if it consists of a plurality of different compounds, so as toreduce the number of different substances per sample and repeat themethod with the subdivisions of the original sample. Depending on thecomplexity of the samples, the steps described above can be performedseveral times, preferably until the sample identified according to themethod of the invention only comprises a limited number of or only onesubstance(s). Preferably said sample comprises substances of similarchemical and/or physical properties, and most preferably said substancesare identical. Preferably, the compound identified according to theabove-described method or its derivative is further formulated in a formsuitable for the application in plant breeding or plant cell and tissueculture.

The compounds which can be tested and identified according to a methodof the invention may be expression libraries, e.g., cDNA expressionlibraries, peptides, proteins, nucleic acids, antibodies, small organiccompounds, hormones, peptidomimetics, PNAs or the like (Milner, NatureMedicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell79 (1994), 193-198 and references cited supra). Said compounds can alsobe functional derivatives or analogues of known inhibitors oractivators. Methods for the preparation of chemical derivatives andanalogues are well known to those skilled in the art and are describedin, for example, Beilstein, Handbook of Organic Chemistry, Springeredition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. andOrganic Synthesis, Wiley, New York, USA. Furthermore, said derivativesand analogues can be tested for their effects according to methods knownin the art. Furthermore, peptidomimetics and/or computer aided design ofappropriate derivatives and analogues can be used, for example,according to the methods described above. The cell or tissue that may beemployed in the method of the invention preferably is a host cell, plantcell or plant tissue of the invention described in the embodimentshereinbefore.

Thus, in a further embodiment the invention relates to a compoundobtained or identified according to the method for identifiying anagonist of the invention said compound being an agonist of thepolypeptide of the present invention or used in the process of thepresent invention.

Accordingly, in one embodiment, the present invention further relates toa compound identified by the method for identifying a compound of thepresent invention.

Said compound is, for example, a homologous of the polypeptide of thepresent invention. Homologues of the polypeptid of the present inventioncan be generated by mutagenesis, e.g., discrete point mutation ortruncation of the polypeptide of the present invenion. As used herein,the term “homologue” refers to a variant form of the protein, which actsas an agonist of the activity of the polypeptide of the presentinvention. An agonist of said protein can retain substantially the same,or a subset, of the biological activities of the polypeptide of thepresent invention. In particular, said agonist confers the increase ofthe expression level of the polypeptide of the present invention and/orthe expression of said agonist in an organisms or part thereof confersthe increase of free and/or bound the fine chemical in the organism orpart thereof.

In one embodiment, the invention relates to an antibody specificallyrecognizing the compound or agonist of the present invention.

The invention also relates to a diagnostic composition comprising atleast one of the aforementioned nucleic acid molecules, vectors,proteins, antibodies or compounds of the invention and optionallysuitable means for detection.

The diagnostic composition of the present invention is suitable for theisolation of mRNA from a cell and contacting the mRNA so obtained with aprobe comprising a nucleic acid probe as described above underhybridizing conditions, detecting the presence of mRNA hybridized to theprobe, and thereby detecting the expression of the protein in the cell.Further methods of detecting the presence of a protein according to thepresent invention comprise immunotechniques well known in the art, forexample enzyme linked immunosorbent assay. Furthermore, it is possibleto use the nucleic acid molecules according to the invention asmolecular markers or primer in plant breeding. Suitable means fordetection are well known to a person skilled in the art, e.g. buffersand solutions for hydridization assays, e.g. the aforementionedsolutions and buffers, further and means for Southern-, Western-,Northern- etc.—blots, as e.g. described in Sambrook et al. are known.

In another embodiment, the present invention relates to a kit comprisingthe nucleic acid molecule, the vector, the host cell, the polypeptide,the antisense nucleic acid, the antibody, plant cell, the plant or planttissue, the harvestable part, the propagation material and/or thecompound or agonist or antagonists identified according to the method ofthe invention.

The compounds of the kit of the present invention may be packaged incontainers such as vials, optionally with/in buffers and/or solution. Ifappropriate, one or more of said components might be packaged in one andthe same container. Additionally or alternatively, one or more of saidcomponents might be adsorbed to a solid support as, e.g. anitrocellulose filter, a glas plate, a chip, or a nylon membrane or tothe well of a micro titerplate. The kit can be used for any of theherein described methods and embodiments, e.g. for the production of thehost cells, transgenic plants, pharmaceutical compositions, detection ofhomologous sequences, identification of antagonists or agonists, as foodor feed or as a supplement thereof, as supplement for the treating ofplants, etc.

Further, the kit can comprise instructions for the use of the kit forany of said embodiments, in particular for the use for producingorganisms or part thereof having an increased free or bound the finechemcial content.

In one embodiment said kit comprises further a nucleic acid moleculeencoding one or more of the aforementioned protein, and/or an antibody,a vector, a host cell, an antisense nucleic acid, a plant cell or planttissue or a plant.

In a further embodiment, the present invention relates to a method forthe production of a agricultural composition providing the nucleic acidmolecule, the vector or the polypeptide of the invention or comprisingthe steps of the method according to the invention for theidentification of said compound, agonist or antagonist; and formulatingthe nucleic acid molecule, the vector or the polypeptide of theinvention or the agonist, or compound identified according to themethods or processes of the present invention or with use of the subjectmatters of the present invention in a form applicable as plantagricultural composition.

In another embodiment, the present invention relates to a method for theproduction of a “the fine chemical”-production supporting plant culturecomposition comprising the steps of the method for of the presentinvention; and formulating the compound identified in a form acceptableas agricultural composition.

Under “acceptable as agricultural composition” is understood, that sucha composition is in agreement with the laws regulating the content offungicides, plant nutrients, herbizides, etc. Preferably such acomposition is without any harm for the protected plants and the animals(humans included) fed therewith.

The present invention also pertains to several embodiments relating tofurther uses and methods. The nucleic acid molecule, polypeptide,protein homologues, fusion proteins, primers, vectors, host cells,described herein can be used in one or more of the following methods:identification of plants useful for fine chemical production asmentioned and related organisms; mapping of genomes; identification andlocalization of sequences of interest; evolutionary studies;determination of regions required for function; modulation of anactivity.

The nucleic acid molecule of the invention, the vector of the inventionor the nucleic acid construct of the invention may also be useful forthe production of organisms resistant to inhibitors of the fine chemicale.g. the amino acid production biosynthesis pathways. In particular, theoverexpression of the polypeptide of the present invention may protectplants against herbicides, which block the amino acid, in particular thefine chemical, synthesis in said plant. Examples of herbicides blockingthe fine chemical synthesis e.g. the amino acid synthesis in plants arefor example sulfonylurea and imidazolinone herbicides which catalyze thefirst step in branched-chain amino acid biosynthesis

Accordingly, the nucleic acid molecules of the present invention have avariety of uses. First, they may be used to identify an organism or aclose relative thereof. Also, they may be used to identify the presencethereof or a relative thereof in a mixed population of microorganisms orplants. By probing the extracted genomic DNA of a culture of a unique ormixed population of plants under stringent conditions with a probespanning a region of the gene of the present invention which is uniqueto this, one can ascertain whether the present invention has been usedor whether it or a close relative is present.

Further, the nucleic acid molecule of the invention may be sufficientlyhomologous to the sequences of related species such that these nucleicacid molecules may serve as markers for the construction of a genomicmap in related organism.

Accordingly, the present invention relates to a method for breedingplants for the production of the fine chemical, comprising

-   (a) providing a first plant variety produced according to the    process of the invention preferably (over)expressing the nucleic    acid molecule of the invention;-   (b) crossing the first plant variety with a second plant variety;    and-   (c) selecting the offspring plants which overproduce the fine    chemical by means of analysis the distribution of a molecular marker    in the offspring representing the first plant variety and its    capability to (over)produce the fine chemical.

Details about the use of molecular markers in breeding can be found inKumar et al., 1999 (Biotech Adv., 17:143-182) and Peleman and van derVoort 2003 (Trends Plant Sci. 2003 July; 8(7):330-334). The molecularmarker can e.g. relate to the nucleic acid molecule of the inventionand/or its expression level. Accordingly, the molecular marker can be aprobe or a PCR primer set useful for identification of the genomicexistence or genomic localisation of the nucleic acid molecule of theinvention, e.g. in a Southern blot analysis or a PCR or its expressionlevel, i.g. in a Northern Blot analysis or a quantitative PCR.Accordingly, in one embodiment, the present invention relates to the useof the nucleic acid molecule of the present invention or encoding thepolypeptide of the present invention as molecular marker for breeding.

The nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. By comparing the sequencesof the invention or used in the process of the invention to thoseencoding similar enzymes from other organisms, the evolutionaryrelatedness of the organisms can be assessed. Similarly, such acomparison permits an assessment of which regions of the sequence areconserved and which are not, which may aid in determining those regionsof the protein which are essential for the functioning of the enzyme.This type of determination is of value for protein engineering studiesand may give an indication of what the protein can tolerate in terms ofmutagenesis without losing function.

Accordingly, the nucleic acid molecule of the invention can be used forthe identification of other nucleic acids conferring an increase of thefine chemical after expression.

Further, the nucleic acid molecule of the invention or a fragment of agene conferring the expression of the polypeptide of the invention,preferably comprising the nucleic acid molecule of the invention, can beused for marker assisted breeding or association mapping of the finechemical derived traits

Accordingly, the nucleic acid of the invention, the polypeptide of theinvention, the nucleic acid construct of the invention, the organisms,the host cell, the microorgansims, the plant, plant tissue, plant cell,or the part thereof of the invention, the vector of the invention, theagonist identified with the method of the invention, the nucleic acidmolecule identified with the method of the present invention, can beused for the production of the fine chemical or of the fine chemical andone or more other ingredients such as amino acids, in particularThreoinine, Alanine, Glutamin, Glutamic acid, Valine, Aspargine,Phenylalanine, Leucine, Proline, Tryptophan Tyrosine, Valine, Isoleucineand Arginine. Accordingly, the nucleic acid of the invention, or thenucleic acid molecule identified with the method of the presentinvention or the complement sequences thereof, the polypeptide of theinvention, the nucleic acid construct of the invention, the organisms,the host cell, the microorgansims, the plant, plant tissue, plant cell,or the part thereof of the invention, the vector of the invention, theantagonist identified with the method of the invention, the antibody ofthe present invention, the antisense molecule of the present invention,can be used for the reduction of the fine chemical in a organism or partthereof, e.g. in a cell.

Further, the nucleic acid of the invention, the polypeptide of theinvention, the nucleic acid construct of the invention, the organisms,the host cell, the microorgansims, the plant, plant tissue, plant cell,or the part thereof of the invention, the vector of the invention, theantagonist or the agonist identified with the method of the invention,the antibody of the presen invention, the antisense molecule of thepresent invention or the nucleic acid molecule identified with themethod of the present invention, can be used for the preparation of anagricultural composition.

Furthermore, the nucleic acid of the invention, the polypeptide of theinvention, the nucleic acid construct of the invention, the organisms,the host cell, the microorgansims, the plant, plant tissue, plant cell,or the part thereof of the invention, the vector of the invention,antagonist or the agonist identified with the method of the invention,the antibody of the presen invention, the antisense molecule of thepresent invention or the nucleic acid molecule identified with themethod of the present invention, can be used for the identification andproduction of compounds capable of conferring a modulation of the finechemical levels in an organism or parts thereof, preferably to identifyand produce compounds conferring an increase of the fine chemical levelsin an organism or parts thereof, if said identified compound is appliedto the organism or part thereof, i.e. as part of its food, or in thegrowing or culture media.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the methods, uses and compounds to be employed inaccordance with the present invention may be retrieved from publiclibraries, using for example electronic devices. For example the publicdatabase “Medline” may be utilized which is available on the Internet,for example under hftp://www.ncbi.nlm.nih.gov/PubMed/medline.html.Further databases and addresses, such as hftp://www.ncbi.nlm.nih.gov/,hftp://www.infobiogen.fr/,hftp://www.fmi.ch/biology/research-tools.html, hftp://www.tigr.org/, areknown to the person skilled in the art and can also be obtained using,e.g., hftp://www.lycos.com. An overview of patent information inbiotechnology and a survey of relevant sources of patent informationuseful for retrospective searching and for current awareness are givenin Berks, TIBTECH 12 (1994), 352-364.

Table 1 gives an overview about the fine chemicals produced by SEQ IDNO: 1 of the present invention. TABLE 1 Fine chemicals produced with SEQID NO: 1 (YNL090W) Metabolite Method Min Max Tryptophane LC 1.26 4.39Proline LC 1.73 6.29 Arginine LC 1.54 4.23 Raffinose LC 2.57 16.05Ferulic Acid LC 1.40 2.41 Phenylalanine LC 1.34 2.75 Tyrosine LC 1.442.55 gamma + beta − Tocopherol LC 0.36 0.66 Cerotic Acid (C26:0) GC 1.413.78 Lignoceric Acid (C24:0) GC 1.34 2.14 Alanine GC 1.21 1.57 GlycineGC 1.46 1.67 Threonine GC 1.21 2.02 Putrescine GC 0.18 0.42 Serine GC1.29 1.96 Valine GC 1.20 2.31 Isoleucine GC 1.30 4.35 Leucine GC 1.515.01 Sinapic Acid GC 1.28 2.08 3,4-Dihydroxyphenylalanine GC 1.37 1.87(=DOPA) Stearic Acid (C18:0) GC 1.16 2.13 beta-Carotene LC 1.77 2.68Column 1 shows the metabolite produced. Column 2 mirrors the analyticmethod and column 3 and 4 shows the minimum and maximum production ofthe respective fine chemical as x-fold.

The present invention is illustrated by the examples, which follow. Thepresent examples illustrate the basic invention without being intendedas limiting the subject of the invention. The content of all of thereferences, patent applications, patents and published patentapplications cited in the present patent application is herewithincorporated by reference.

EXAMPLES Example 1 Cloning SEQ ID NO: 1 in Escherichia coli

SEQ ID NO: 1 was cloned into the plasmids pBR322 (Sutcliffe, J. G.(1979) Proc. Natl Acad. Sci. USA, 75: 3737-3741); pACYC177 (Change &Cohen (1978) J. Bacteriol. 134: 1141-1156); plasmids of the pBS series(pBSSK+, pBSSK− and others; Stratagene, LaJolla, USA) or cosmids such asSuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T. J.Rosenthal, A., and Waterson, R. H. (1987) Gene 53: 283-286) forexpression in E. coli using known, well-established procedures (see, forexample, Sambrook, J. et al. (1989) “Molecular Cloning: A LaboratoryManual”. Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al.(1994) “Current Protocols in Molecular Biology”, John Wiley & Sons).

Example 2 DNA Sequencing and Computerized Functional Analysis

The DNA was sequenced by standard procedures, in particular the chaindetermination method, using ABI377 sequencers (see, for example,Fleischman, R. D. et al. (1995) “Whole-genome Random Sequencing andAssembly of Haemophilus Influenzae Rd., Science 269; 496-512)”.

Example 3 In-Vivo Mutagenesis

An in vivo mutagenesis of Corynebacterium glutamicum for the productionof amino acids can be carried out by passing a plasmid DNA (or anothervector DNA) through E. coli and other microorganisms (for exampleBacillus spp. or yeasts such as Saccharomyces cerevisiae), which are notcapable of maintaining the integrity of its genetic information. Usualmutator strains have mutations in the genes for the DNA repair system[for example mutHLS, mutD, mutT and the like; for comparison, see Rupp,W. D. (1996) DNA repair mechanisms in Escherichia coli and Salmonella,pp. 2277-2294, ASM: Washington]. The skilled worker knows these strains.The use of these strains is illustrated for example in Greener, A. andCallahan, M. (1994) Strategies 7; 32-34.

Example 4 DNA Transfer between Escherichia coli and CorynebacteriumGlutamicum

Several Corynebacterium and Brevibacterium species comprise endogenousplasmids (such as, for example, pHM1519 or pBL1), which replicateautonomously (for a review, see, for example, Martin, J. F. et al.(1987) Biotechnology 5: 137-146). Shuttle vectors for Escherichia coliand Corynebacterium glutamicum can be constructed easily using standardvectors for E. coli (Sambrook, J. et al., (1989), “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons), which have a replication origin for, and suitable marker from,Corynebacterium glutamicum added. Such replication origins arepreferably taken from endogenous plasmids, which have been isolated fromCorynebacterium and Brevibacterium species. Genes, which are used inparticular as transformation markers for these species are genes forkanamycin resistance (such as those which originate from the Tn5 orTn-903 transposon) or for chloramphenicol resistance (Winnacker, E. L.(1987) “From Genes to Clones—Introduction to Gene Technology, VCH,Weinheim). There are many examples in the literature of the preparationof a large multiplicity of shuttle vectors which are replicated in E.coli and C. glutamicum and which can be used for various purposesincluding the overexpression of genes (see, for example, Yoshihama, M.et al. (1985) J. Bacteriol. 162: 591-597, Martin, J. F. et al., (1987)Biotechnology, 5: 137-146 and Eikmanns, B. J. et al. (1992) Gene 102:93-98). Suitable vectors which replicate in coryneform bacteria are forexample, pZ1 (Menkel et al., Appl. Environ. Microbiol., 64, 1989:549-554) pEkEx1 (Eikmanns et al., Gene 102, 1991: 93-98) or pHS2-1(Sonnen et al, Gene 107, 1991: 69-74). These vectors are based on thecryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors such as,for example, those based on pCG4 (U.S. Pat. No. 4,489,160), pNG2(Serwold-Davis et al., FEMS Microbiol. Lett., 66, 1990: 119-124) or pAG1(U.S. Pat. No. 5,158,891) can be used in the same manner.

Using standard methods, it is possible to clone a gene of interest intoone of the above-described shuttle vectors and to introduce such hybridvectors into Corynebacterium glutamicum strains. The transformation ofC. glutamicum can be achieved by protoplast transformation (Kastsumata,R. et al., (1984) J. Bacteriol. 159, 306-311), electroporation (Liebl,E. et al., (1989) FEMS Microbiol. Letters, 53: 399-303) and in thosecases where specific vectors are used also by conjugation (such as, forexample, described in Schäfer, A., et al. (1990) J. Bacteriol. 172:1663-1666). Likewise, it is possible to transfer the shuttle vectors forC. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum(using standard methods known in the art) and transforming it into E.coli. This transformation step can be carried out using standardmethods, but preferably using a Mcr-deficient E. coli strain, such asNM522 (Gough & Murray (1983) J. Mol. Biol. 166: 1-19).

If the transformed sequence(s) is/are to be integrated advantageouslyinto the genome of the coryneform bacteria, standard techniques known tothe skilled worker also exist for this purpose. Examples, which are usedfor this purpose are plasmid vectors as they have been described byRemscheid et al. (Appl. Environ. Microbiol., 60, 1994: 126-132) for theduplication and amplification of the hom-thrB operon. In this method,the complete gene is cloned into a plasmid vector, which is capable ofreplication in a host such as E. coli, but not in C. glutamicum.Suitable vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 1983: 784-791), pKIBmob or pK19mob (Schäfer et al., Gene 145, 1994:69-73), pGEM-T (Promega Corp., Madison, Wis., USA), pCR2.1-TOPO(Schuman, J. Biol. Chem., 269, 1994: 32678-32684, U.S. Pat. No.5,487,993), pCR®Blunt (Invitrogen, Groningen, the Netherlands) or pEM1(Schrumpf et al., J. Bacteriol., 173, 1991: 4510-4516).

Example 5 Determining the Expression of the Mutant/Transgenic Protein

The observations of the acivity of a mutated, or transgenic, protein ina transformed host cell are based on the fact that the protein isexpressed in a similar manner and in a similar quantity as the wild-typeprotein. A suitable method for determining the transcription quantity ofthe mutant, or transgenic, gene (a sign for the amount of mRNA which isavailable for the translation of the gene product) is to carry out aNorthern blot (see, for example, Ausubel et al., (1988) CurrentProtocols in Molecular Biology, Wiley: New York), where a primer whichis designed in such a way that it binds to the gene of interest isprovided with a detectable marker (usually a radioactive orchemiluminescent marker) so that, when the total RNA of a culture of theorganism is extracted, separated on a gel, applied to a stable matrixand incubated with this probe, the binding and quantity of the bindingof the probe indicates the presence and also the amount of mRNA for thisgene. Another method is a quantitative PCR. This information detects theextent to which the gene has been transcribed. Total cell RNA can beisolated from Corynebacterium glutamicum by a variety of methods, whichare known in the art, as described in Bormann, E. R. et al., (1992) Mol.Microbiol. 6: 317-326.

Standard techniques, such as Western blot, may be employed to determinethe presence or relative amount of protein translated from this mRNA(see, for example, Ausubel et al. (1988) “Current Protocols in MolecularBiology”, Wiley, New York). In this method, total cell proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose and incubated with a probe, such as an antibody,which binds specifically to the desired protein. This probe is usuallyprovided directly or indirectly with a chemiluminescent or calorimetricmarker, which can be detected readily. The presence and the observedamount of marker indicate the presence and the amount of the soughtmutant protein in the cell. However, other methods are also known.

Example 6 Growth of Genetically Modified Corynebacterium glutamicum:Media and Culture Conditions

Genetically modified Corynebacteria are grown in synthetic or naturalgrowth media. A number of different growth media for Corynebacteria areknown and widely available (Lieb et al. (1989) Appl. Microbiol.Biotechnol. 32: 205-210; von der Osten et al. (1998) BiotechnologyLetters 11: 11-16; Patent DE 4 120 867; Liebl (1992) “The GenusCorynebacterium”, in: The Procaryotes, Vol. II, Balows, A., et al., Ed.Springer-Verlag).

Said media which can be used according to the invention usually consistof one or more carbon sources, nitrogen sources, inorganic salts,vitamins and trace elements. Preferred carbon sources are sugars such asmono-, di- or polysaccharides. Examples of very good carbon sources areglucose, fructose, mannose, galactose, ribose, sorbose, ribulose,lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars mayalso be added to the media via complex compounds such as molasses orother by-products of sugar refining. It may also be advantageous to addmixtures of various carbon sources. Other possible carbon sources arealcohols and/or organic acids such as methanol, ethanol, acetic acid orlactic acid. Nitrogen sources are usually organic or inorganic nitrogencompounds or materials containing said compounds. Examples of nitrogensources include ammonia gas, aqueous ammonia solutions or ammonium saltssuch as NH₄Cl, or (NH₄)₂SO₄, NH₄OH, nitrates, urea, amino acids orcomplex nitrogen sources such as cornsteep liquor, soybean flour,soybean protein, yeast extract, meat extract and others. Mixtures of theabove nitrogen sources may be used advantageously.

Inorganic salt compounds, which may be included in the media comprisethe chloride, phosphorus or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents includedihydroxyphenols such as catechol or protocatechulate or organic acidssuch as citric acid. The media usually also contain other growth factorssuch as vitamins or growth promoters, which include, for example,biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenateand pyridoxine. Growth factors and salts are frequently derived fromcomplex media components such as yeast extract, molasses, cornsteepliquor and the like. The exact composition of the compounds used in themedia depends heavily on the particular experiment and is decided uponindividually for each specific case. Information on the optimization ofmedia can be found in the textbook “Applied Microbiol. Physiology, APractical Approach” (Ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997)S. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained fromcommercial suppliers, for example Standard 1 (Merck) or BHI (Brain heartinfusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barund 121° C.) or by filter sterilization. The components may besterilized either together or, if required, separately. All mediacomponents may be present at the start of the cultivation or addedcontinuously or batchwise, as desired.

The culture conditions are defined separately for each experiment. Thetemperature is normally between 15° C. and 45° C. and may be keptconstant or may be altered during the experiment. The pH of the mediumshould be in the range from 5 to 8.5, preferably around 7.0, and can bemaintained by adding buffers to the media. An example of a buffer forthis purpose is a potassium phosphate buffer. Synthetic buffers such asMOPS, HEPES, ACES and the like may be used as an alternative orsimultaneously. The culture pH value may also be kept constant duringthe culture period by addition of, for example, NaOH or NH₄OH. Ifcomplex media components such as yeast extract are used, additionalbuffers are required less since many complex compounds have a highbuffer capacity. When using a fermenter for the culture ofmicroorganisms, the pH value can also be regulated using gaseousammonia.

The incubation period is generally in a range of from several hours toseveral days. This time period is selected in such a way that themaximum amount of product accumulates in the fermentation broth. Thegrowth experiments, which are disclosed can be carried out in amultiplicity of containers such as microtiter plates, glass tubes, glassflasks or glass or metal fermenters of various sizes. To screen a largenumber of clones, the microorganisms should be grown in microtiterplates, glass tubes or shake flasks, either using simple flasks orbaffle flasks. 100 ml shake flasks filled with 10% (based on the volume)of the growth medium required are preferably used. The flasks should beshaken on an orbital shaker (amplitude 25 mm) at a rate ranging from 100to 300 rpm. Evaporation losses can be reduced by maintaining a humidatmosphere; as an alternative, a mathematical correction should becarried out for the evaporation losses.

If genetically modified clones are examined, an unmodified controlclone, or a control clone, which contains the basic plasmid withoutinsertion, should also be included in the tests. If a transgenicsequence is expressed, a control clone should advantageously again beincluded in these tests. The medium is advantageously inoculated to anOD600 of 0.5 to 1.5 using cells which have been grown on agar plates,such as CM plates (10 g/l glucose, 2.5 g/l NaCl, 2 g/l urea, 10 g/lpolypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/l agar, pHvalue 6.8 established with 2M NaOH), which have been incubated at 30° C.The media are inoculated either by introducing a saline solution of C.glutamicum cells from CM plates or by addition of a liquid preculture ofthis bacterium.

Example 7 In-Vitro Analysis of the Function of the Proteins Encoded bythe Transformed Sequences

The determination of the activities and kinetic parameters of enzymes iswell known in the art. Experiments for determining the activity of aspecific modified enzyme must be adapted to the specific activity of thewild-enzyme type, which is well within the capabilities of the skilledworker. Overviews of enzymes in general and specific details regardingthe structure, kinetics, principles, methods, applications and examplesfor the determination of many enzyme activities can be found for examplein the following literature: Dixon, M., and Webb, E. C: (1979) Enzymes,Longmans, London; Fersht (1985) Enzyme Structure and Mechanism, Freeman,New York; Walsh (1979) Enzymatic Reaction Mechanisms. Freeman, SanFrancisco; Price, N. C., Stevens, L. (1982) Fundamentals of Enzymology.Oxford Univ. Press: Oxford; Boyer, P. D: Ed. (1983) The Enzymes, 3rd Ed.Academic Press, New York; Bisswanger, H. (1994) Enzymkinetik, 2nd Ed.VCH, Weinheim (ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., Graβl,M. Ed. (1983-1986) Methods of Enzymatic Analysis, 3rd Ed. Vol. I-XII,Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of IndustrialChemistry (1987) Vol. A9, “Enzymes”, VCH, Weinheim, pp. 352-363.

Example 8 Analysis of the Effect of the Nucleic Acid Molecule on theProduction of the Amino Acids

The effect of the genetic modification in C. glutamicum on theproduction of an amino acid can be determined by growing the modifiedmicroorganisms under suitable conditions (such as those described above)and analyzing the medium and/or the cellular components for theincreased production of the amino acid. Such analytical techniques arewell known to the skilled worker and encompass spectroscopy, thin-layerchromatography, various types of staining methods, enzymatic andmicrobiological methods and analytical chromatography such ashigh-performance liquid chromatography (see, for example, Ullman,Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp.443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) “Applicationsof HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3,Chapter III: “Product recovery and purification”, pp. 469-714, VCH:Weinheim; Belter, P. A. et al. (1988) Bioseparations: downstreamprocessing for Biotechnology, John Wiley and Sons; Kennedy, J. F. andCabral, J. M. S. (1992) Recovery processes for biological Materials,John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D. (1988)Biochemical Separations, in Ullmann's Encyclopedia of IndustrialChemistry, Vol. B3; chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to the determination of the fermentation end product, othercomponents of the metabolic pathways which are used for the productionof the desired compound, such as intermediates and by-products, may alsobe analyzed in order to determine the total productivity of theorganism, the yield and/or production efficiency of the compound. Theanalytical methods encompass determining the amounts of nutrients in themedium (for example sugars, hydrocarbons, nitrogen sources, phosphateand other ions), determining biomass composition and growth, analyzingthe production of ordinary metabolites from biosynthetic pathways andmeasuring gases generated during the fermentation. Standard methods forthese are described in Applied Microbial Physiology; A PracticalApproach, P. M. Rhodes and P. F. Stanbury, Ed. IRL Press, pp. 103-129;131-163 and 165-192 (ISBN: 0199635773) and the references cited therein.

Example 9 Purification of the Amino Acid

The amino acid can be recovered from cells or from the supernatant ofthe above-described culture by a variety of methods known in the art.For example, the culture supernatant is recovered first. To this end,the cells are harvested from the culture by slow centrifugation. Cellscan generally be disrupted or lysed by standard techniques such asmechanical force or sonication. The cell debris is removed bycentrifugation and the supernatant fraction, if appropriate togetherwith the culture supernatant, is used for the further purification ofthe amino acid. However, it is also possible to process the supernatantalone if the amino acid is present in the supernatant in sufficientlyhigh a concentration. In this case, the amino acid, or the amino acidmixture, can be purified further for example via extraction and/or saltprecipitation or via ion-exchange chromatography.

If required and desired, further chromatography steps with a suitableresin may follow, the amino acid, but not many contaminants in thesample, being retained on the chromatography resin or the contaminants,but not the sample with the product (amino acid), being retained on theresin. If necessary, these chromatography steps may be repeated, usingidentical or other chromatography resins. The skilled worker is familiarwith the selection of suitable chromatography resin and the mosteffective use for a particular molecule to be purified. The purifiedproduct can be concentrated by filtration or ultrafiltration and storedat a temperature at which maximum product stability is ensured. Manypurification methods, which are not limited to the above purificationmethod are known in the art. They are described, for example, in Bailey,J. E. & Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill:New York (1986).

Identity and purity of the amino acid isolated can be determined bystandard techniques of the art. They encompass high-performance liquidchromatography (HPLC), spectroscopic methods, mass spectrometry (MS),staining methods, thin-layer chromatography, NIRS, enzyme assay ormicrobiological assays. These analytical methods are compiled in: Pateket al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al.(1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) BioprocessEngineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry(1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp.559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons;Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

Example 10 Cloning SEQ ID NO: 1 for the Expression in Plants

Unless otherwise specified, standard methods as described in Sambrook etal., Molecular Cloning: A laboratory manual, Cold Spring Harbor 1989,Cold Spring Harbor Laboratory Press are used.

SEQ ID NO: 1 is amplified by PCR as described in the protocol of the PfuTurbo or DNA Herculase polymerase (Stratagene).

The composition for the protocol of the Pfu Turbo DNA polymerase was asfollows: 1×PCR buffer (Stratagene), 0.2 mM of each dNTP, 100 ng genomicDNA of Saccharomyces cerevisiae (strain S288C; Research Genetics, Inc.,now Invitrogen) or Escherichia coli (strain MG1655; E. coli GeneticStock Center), 50 pmol forward primer, 50 pmol reverse primer, 2.5 u PfuTurbo DNA polymerase. The amplification cycles were as follows:

-   1 cycle of 3 minutes at 94-95° C., followed by 25-36 cycles of in    each case 1 minute at 95° C. or 30 seconds at 94° C., 45 seconds at    50° C., 30 seconds at 50° C. or 30 seconds at 55° C. and 210-480    seconds at 72° C., followed by 1 cycle of 8 minutes at 72° C., then    4° C.

The composition for the protocol of the Herculase polymerase was asfollows: 1×PCR buffer (Stratagene), 0.2 mM of each dNTP, 100 ng genomicDNA of Saccharomyces cerevisiae (strain S288C; Research Genetics, Inc.,now Invitrogen) or Escherichia coli (strain MG1655; E. coli GeneticStock Center), 50 pmol forward primer, 50 pmol reverse primer, 2.5 uHerculase polymerase. The amplification cycles were as follows:

1 cycle of 2-3 minutes at 94° C., followed by 25-30 cycles of in eachcase 30 seconds at 94° C., 30 seconds at 55-60° C. and 5-10 minutes at72° C., followed by 1 cycle of 10 minutes at 72° C., then 4° C.

The following primer sequences were selected for the gene SEQ ID NO: 1:i) forward primer (SEQ ID NO: 53) 5′-ATGTCTGAAAAGGCCGTTAGAAGG-3′ ii)reverse primer (SEQ ID NO: 54) 5′-TTATAAAATTATGCAACAGTTAGCCC-3′

Thereafter, the amplificate was purified over QlAquick columns followingthe standard protocol (Qiagen).

For the cloning of PCR-products, produced by Pfu Turbo DNA polymerase,the vector DNA (30 ng) was restricted with SmaI following the standardprotocol (MBI Fermentas) and stopped by addition of high-salt buffer.The restricted vector fragments were purified via Nucleobond columnsusing the standard protocol (Macherey-Nagel). Thereafter, the linearizedvector was dephosphorylated following the standard protocol (MBIFermentas).

The PCR-products, produced by Pfu Turbo DNA polymerase, were directlycloned into the processed binary vector.

The DNA termini of the PCR-products, produced by Herculase DNApolymerase, were blunted in a second synthesis reaction using Pfu TurboDNA polymerase. The composition for the protocol of the blunting theDNA-termini was as follows: 0.2 mM blunting dTTP and 1.25 u Pfu TurboDNA polymerase. The reaction was incubated at 72° C. for 30 minutes.Then the PCR-products were cloned into the processed vector as well.

A binary vector comprising a selection cassette (promoter, selectionmarker, terminator) and an expression cassette with promoter, cloningcassette and terminator sequence between the T-DNA border sequences wasused. In addition to those within the cloning cassette, the binaryvector has no SmaI cleavage site. Binary vectors which can be used areknown to the skilled worker; an overview of binary vectors and their usecan be found in Hellens, R., Mullineaux, P. and Klee H., [(2000) “Aguide to Agrobacterium binary vectors”, Trends in Plant Science, Vol. 5No. 10, 446-451. Depending on the vector used, cloning mayadvantageously also be carried out via other restriction enzymes.Suitable advantageous cleavage sites can be added to the ORF by usingsuitable primers for the PCR amplification.

Approximately 30 ng of prepared vector and a defined amount of preparedamplificate were mixed and ligated by addition of ligase.

The ligated vectors were transformed in the same reaction vessel byaddition of competent E. coli cells (strain DH5alpha) and incubation for20 minutes at 1° C. followed by a heat shock for 90 seconds at 42° C.and cooling to 4° C. Then, complete medium (SOC) was added and themixture was incubated for 45 minutes at 37° C. The entire mixture wassubsequently plated onto an agar plate with antibiotics (selected as afunction of the binary vector used) and incubated overnight at 37° C.

The outcome of the cloning step was verified by amplification with theaid of primers which bind upstream and downstream of the integrationsite, thus allowing the amplification of the insertion. In additioncombinations of the above mentioned gene specific primers and upstreamand downstream primers were used in PCR reactions to identify cloneswith the correct insert orientation. The amplifications were carried asdescribed in the protocol of Taq DNA polymerase (Gibco-BRL).

The amplification cycles were as follows: 1 cycle of 5 minutes at 94°C., followed by 35 cycles of in each case 15 seconds at 94° C., 15seconds at 50-66° C. and 5 minutes at 72° C., followed by 1 cycle of 10minutes at 72° C., then 4° C.

Several colonies were checked, but only one colony for which a PCRproduct of the expected size was detected was used in the followingsteps.

A portion of this positive colony was transferred into a reaction vesselfilled with complete medium (LB) and incubated overnight at 37° C. TheLB medium contained an antibiotic chosen to suit the binary vector (seeabove) used and the resistance gene present therein in order to selectthe clone.

The plasmid preparation was carried out as specified in the Qiaprepstandard protocol (Qiagen).

Example 11 Generation of Transgenic Plants which Express SEQ ID NO: 1

1 ng of the plasmid DNA isolated was transformed by electroporation intocompetent cells of Agrobacterium tumefaciens, of strain GV 3101 pMP90(Koncz and Schell, Mol. Gen. Gent. 204, 383-396, 1986). The choice ofthe agrobacterial strain depends on the choice of the binary vector. Anoverview of possible strains and their properties is found in Hellens,R., Mullineaux, P. and Klee H., (2000)” A guide to Agrobacterium binaryvectors, Trends in Plant Science, Vol. 5 No. 10, 446-451. Thereafter,complete medium (YEP) was added and the mixture was transferred into afresh reaction vessel for 3 hours at 28° C. Thereafter, all of thereaction mixture was plated onto YEP agar plates supplemented with therespective antibiotics, for example rifampicin and gentamycin for GV3101pMP90, and a further antibiotic for the selection onto the binaryvector, was plated, and incubated for 48 hours at 28° C.

The agrobacteria generated in Example 10, which contains the plasmidconstruct were then used for the transformation of plants.

A colony was picked from the agar plate with the aid of a pipette tipand taken up in 3 ml of liquid TB medium, which also contained suitableantibiotics, depending on the agrobacterial strain and the binaryplasmid. The preculture was grown for 48 hours at 28° C. and 120 rpm.

400 ml of LB medium containing the same antibiotics as above were usedfor the main culture. The preculture was transferred into the mainculture. It was grown for 18 hours at 28° C. and 120 rpm. Aftercentrifugation at 4000 rpm, the pellet was resuspended in infiltrationmedium (MS medium, 10% sucrose).

In order to grow the plants for the transformation, dishes (Piki Saat80, green, provided with a screen bottom, 30×20×4.5 cm, fromWiesauplast, Kunststofftechnik, Germany) were half-filled with a GS 90substrate (standard soil, Werkverband E.V., Germany). The dishes werewatered overnight with 0.05% Proplant solution (Chimac-Apriphar,Belgium). Arabidopsis thaliana C24 seeds (Nottingham Arabidopsis StockCentre, UK; NASC Stock N906) were scattered over the dish, approximately1000 seeds per dish. The dishes were covered with a hood and placed inthe stratification facility (8 h, 110μ μmol/m²/s⁻¹, 22° C.; 16 h, dark,6° C.). After 5 days, the dishes were placed into the short-daycontrolled environment chamber (8 h 130 μmol/m²/s⁻¹, 22° C.; 16 h, dark20° C.), where they remained for approximately 10 days until the firsttrue leaves had formed.

The seedlings were transferred into pots containing the same substrate(Teku pots, 7 cm, LC series, manufactured by Pöppelmann GmbH & Co,Germany). Five plants were pricked out into each pot. The pots were thenreturned into the short-day controlled environment chamber for the plantto continue growing.

After 10 days, the plants were transferred into the greenhouse cabinet(supplementary illumination, 16 h, 340 μE, 22° C.; 8 h, dark, 20° C.),where they were allowed to grow for further 17 days.

For the transformation, 6-week-old Arabidopsis plants which had juststarted flowering were immersed for 10 seconds into the above-describedagrobacterial suspension which had previously been treated with 10 μlSilwett L77 (Crompton S.A., Osi Specialties, Switzerland). The method inquestion is described in Clough and Bent, 1998 (Clough, J C and Bent, AF. 1998 Floral dip: a simplified method for Agrobacterium-mediatedtransformation of Arabidopsis thaliana, Plant J. 16:735-743.

The plants were subsequently placed for 18 hours into a humid chamber.Thereafter, the pots were returned to the greenhouse for the plants tocontinue growing. The plants remained in the greenhouse for another 10weeks until the seeds were ready for harvesting.

Depending on the resistance marker used for the selection of thetransformed plantsthe harvested seeds were planted in the greenhouse andsubjected to a spray selection or else first sterilized and then grownon agar plates supplemented with the respective selection agent. In caseof BASTA®-resistance, plantlets were sprayed four times at an intervalof 2 to 3 days with 0.02% BASTA® and transformed plants were allowed toset seeds. The seeds of the transgenic A. thaliana plants were stored inthe freezer (at −20° C.).

Example 12 Plant Culture for Bioanalytical Analyses

For the bioanalytical analyses of the transgenic plants, the latter weregrown uniformly a specific culture facility. To this end the GS-90substrate as the compost mixture was introduced into the potting machine(Laible System GmbH, Singen, Germany) and filled into the pots.Thereafter, 35 pots were combined in one dish and treated with Previcur.For the treatment, 25 ml of Previcur were taken up in 10 l of tap water.This amount was sufficient for the treatment of approximately 200 pots.The pots were placed into the Previcur solution and additionallyirrigated overhead with tap water without Previcur. They were usedwithin four days.

For the sowing, the seeds, which had been stored in the refrigerator (at−20° C.), were removed from the Eppendorf tubes with the aid of atoothpick and transferred into the pots with the compost. In total,approximately 5 to 12 seeds were distributed in the middle of the pot.

After the seeds had been sown, the dishes with the pots were coveredwith matching plastic hood and placed into the stratification chamberfor 4 days in the dark at 4° C. The humidity was approximately 90%.After the stratification, the test plants were grown for 22 to 23 daysat a 16-h-light, 8-h-dark rhythm at 20° C., an atmospheric humidity of60% and a CO₂ concentration of approximately 400 ppm. The light sourcesused were Powerstar HQI-T 250 W/D Daylight lamps from Osram, whichgenerate a light resembling the solar color spectrum with a lightintensity of approximately 220 μE/m2/s-1.

When the plants were 8, 9 and 10 days old, they were subjected toselection for the resistance marker Approximately 1400 pots withtransgenic plants were treated with 1 l 0.015% vol/vol of Basta®(Glufosinate-ammonium) solution in water (Aventis Cropsience,GermanyAfter a further 3 to 4 days, the transgenic, resistant seedlings(plantlets in the 4-leaf stage) could be distinguished clearly from theuntransformed plantlets. The nontransgenic seedlings were bleached ordead. The transgenic resistance plants were thinned when they hadreached the age of 14 days. The plants, which had grown best in thecenter of the pot were considered the target plants. All the remainingplants were removed carefully with the aid of metal tweezers anddiscarded.

During their growth, the plants received overhead irrigation withdistilled water (onto the compost) and bottom irrigation into theplacement grooves. Once the grown plants had reached the age of 23 days,they were harvested.

Example 13 Metabolic Analysis of Transformed Plants

The modifications identified in accordance with the invention, in thecontent of above-described metabolites, were identified by the followingprocedure.

a) Sampling and Storage of the Samples

Sampling was performed directly in the controlled-environment chamber.The plants were cut using small laboratory scissors, rapidly weighed onlaboratory scales, transferred into a pre-cooled extraction sleeve andplaced into an aluminum rack cooled by liquid nitrogen. If required, theextraction sleeves can be stored in the freezer at −80° C. The timeelapsing between cutting the plant to freezing it in liquid nitrogenamounted to not more than 10 to 20 seconds.

b) Lyophilization

During the experiment, care was taken that the plants either remained inthe deep-frozen state (temperatures<−40° C.) or were freed from water bylyophilization until the first contact with solvents.

The aluminum rack with the plant samples in the extraction sleeves wasplaced into the pre-cooled (−40° C.) lyophilization facility. Theinitial temperature during the main drying phase was −35° C. and thepressure was 0.120 mbar. During the drying phase, the parameters werealtered following a pressure and temperature program. The finaltemperature after 12 hours was +30° C. and the final pressure was 0.001to 0.004 mbar. After the vacuum pump and the refrigerating machine hadbeen switched off, the system was flushed with air (dried via a dryingtube) or argon.

c) Extraction

Immediately after the lyophilization apparatus had been flushed, theextraction sleeves with the lyophilized plant material were transferredinto the 5 ml extraction cartridges of the ASE device (AcceleratedSolvent Extractor ASE 200 with Solvent Controller and AutoASE software(DIONEX)).

The 24 sample positions of an ASE device (Accelerated Solvent ExtractorASE 200 with Solvent Controller and AutoASE software (DIONEX)) werefilled with plant samples, including some samples for testing qualitycontrol.

The polar substances were extracted with approximately 10 ml ofmethanol/water (80/20, v/v) at T=70° C. and p=140 bar, 5 minutesheating-up phase, 1 minute static extraction. The more lipophilicsubstances were extracted with approximately 10 ml ofmethanol/dichloromethane (40/60, v/v) at T=70° C. and p=140 bar, 5minute heating-up phase, 1 minute static extraction. The two solventmixtures were extracted into the same glass tubes (centrifuge tubes, 50ml, equipped with screw cap and pierceable septum for the ASE (DIONEX)).

The solution was treated with internal standards: ribitol,L-glycine-2,2-d₂, L-alanine-2,3,3,3-d₄, methionine-methyl-d₃, andα-methylglucopyranoside and methyl nonadecanoate, methyl undecanoate,methyl tridecanoate, methyl pentadecanoate, methyl nonacosanoate.

The total extract was treated with 8 ml of water. The solid residue ofthe plant sample and the extraction sleeve were discarded.

The extract was shaken and then centrifuged for 5 to 10 minutes at atleast 1400 g in order to accelerate phase separation. 1 ml of thesupernatant methanol/water phase (“polar phase”, colorless) was removedfor the further GC analysis, and 1 ml was removed for the LC analysis.The remainder of the methanol/water phase was discarded. 0.5 ml of theorganic phase (“lipid phase”, dark green) was removed for the further GCanalysis and 0.5 ml was removed for the LC analysis. All the portionsremoved were evaporated to dryness using the IR Dancer infrared vacuumevaporator (Hettich). The maximum temperature during the evaporationprocess did not exceed 40° C. Pressure in the apparatus was not lessthan 10 mbar.

d) Processing the Lipid Phase for the LC/MS or LC/MS/MS Analysis

The lipid extract, which had been evaporated to dryness was taken up inmobile phase. The HPLC was run with gradient elution.

The polar extract, which had been evaporated to dryness was taken up inmobile phase. The HPLC was run with gradient elution.

e) Derivatization of the Lipid Phase for the GC/MS Analysis

For the transmethanolysis, a mixture of 140 μl of chloroform, 37 μl ofhydrochloric acid (37% by weight HCl in water), 320 μl of methanol and20 μl of toluene was added to the evaporated extract. The vessel wassealed tightly and heated for 2 hours at 100° C., with shaking. Thesolution was subsequently evaporated to dryness. The residue was driedcompletely.

The methoximation of the carbonyl groups was carried out by reactionwith methoxyamine hydrochloride (5 mg/ml in pyridine, 100 μl for 1.5hours at 60° C.) in a tightly sealed vessel. 20 μl of a solution ofodd-numbered, straight-chain fatty acids (solution of each 0.3 mg/mL offatty acids from 7 to 25 carbon atoms and each 0.6 mg/mL of fatty acidswith 27, 29 and 31 carbon atoms in 3/7 (v/v) pyridine/toluene) wereadded as time standards. Finally, the derivatization with 100 μl ofN-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was carriedout for 30 minutes at 60° C., again in the tightly sealed vessel. Thefinal volume before injection into the GC was 220 μl.

f) Derivatization of the Polar Phase for the GC/MS Analysis

The methoximation of the carbonyl groups was carried out by reactionwith methoxyamine hydrochloride (5 mg/ml in pyridine, 50 μl for 1.5hours at 60° C.) in a tightly sealed vessel. 10 μl of a solution ofodd-numbered, straight-chain fatty acids (solution of each 0.3 mg/mL offatty acids from 7 to 25 carbon atoms and each 0.6 mg/mL of fatty acidswith 27, 29 and 31 carbon atoms in 3/7 (v/v) pyridine/toluene) wereadded as time standards. Finally, the derivatization with 50 μl ofN-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was carriedout for 30 minutes at 60° C., again in the tightly sealed vessel. Thefinal volume before injection into the GC was 110 μl.

g) Analysis of the Various Plant Samples

The samples were measured in individual series of 20 plant samples each(also referred to as sequences), each sequence containing at least 5wild-type plants as controls. The peak area of each analyte was dividedby the peak area of the respective internal standard. The data werestandardized for the fresh weight established for the plant. The valuescalculated thus were related to the wild-type control group by beingdivided by the mean of the corresponding data of the wild-type controlgroup of the same sequence. The values obtained were referred to asratio_by_WT, they are comparable between sequences and indicate how muchthe analyte concentration in the mutant differs in relation to thewild-type control. Appropiate controls were done before to proof thatthe vector and transformation procedure itself has no significantinfluence on the metabolic composition of the plants. Therefore thedesribed changes in comparison with wildtypes were caused by theintroduced genes.

As an alternative, the amino acids can be detected advantageously viaHPLC separation in ethanolic extract as described by Geigenberger et al.(Plant Cell & Environ, 19, 1996: 43-55).

The results of the different plant analyses can be seen from the table1.

Column 1 in Table 1 shows the amino acid analyzed. Columns 3 and 4 showsthe ratio of the analyzed amino acid between the transgenic plants andthe wild type; Increase of the metabolites: Max: maximal x-fold(normalised to wild type)−Min: minimal x-fold (normalised to wild type).Decrease of the metabolites: Max: maximal x-fold (normalised to wildtype) (minimal decrease), Min: minimal x-fold (normalised to wild type)(maximal decrease). Column 2 indicates the analytical method.

When the analyses were repeated independently, all results proved to besignificant.

Example 14a Engineering Ryegrass Plants by Over-Expressing YNL090W, e.g.from Saccharomyces cerevisiae or Plants

Seeds of several different ryegrass varieties can be used as explantsources for transformation, including the commercial variety Gunneavailable from Svalof Weibull seed company or the variety Affinity.Seeds are surface-sterilized sequentially with 1% Tween-20 for 1 minute,100% bleach for 60 minutes, 3 rinses with 5 minutes each with de-ionizedand distilled H2O, and then germinated for 3-4 days on moist, sterilefilter paper in the dark. Seedlings are further sterilized for 1 minutewith 1% Tween-20, 5 minutes with 75% bleach, and rinsed 3 times withddH₂O, 5 min each.

Surface-sterilized seeds are placed on the callus induction mediumcontaining Murashige and Skoog basal salts and vitamins, 20 g/l sucrose,150 mg/l asparagine, 500 mg/l casein hydrolysate, 3 g/l Phytagel, 10mg/l BAP, and 5 mg/l dicamba. Plates are incubated in the dark at 25° C.for 4 weeks for seed germination and embryogenic callus induction.

After 4 weeks on the callus induction medium, the shoots and roots ofthe seedlings are trimmed away, the callus is transferred to freshmedia, is maintained in culture for another 4 weeks, and is thentransferred to MSO medium in light for 2 weeks. Several pieces of callus(11-17 weeks old) are either strained through a 10 mesh sieve and putonto callus induction medium, or are cultured in 100 ml of liquidryegrass callus induction media (same medium as for callus inductionwith agar) in a 250 ml flask. The flask is wrapped in foil and shaken at175 rpm in the dark at 23° C. for 1 week. Sieving the liquid culturewith a 40-mesh sieve is collected the cells. The fraction collected onthe sieve is plated and is cultured on solid ryegrass callus inductionmedium for 1 week in the dark at 25° C. The callus is then transferredto and is cultured on MS medium containing 1% sucrose for 2 weeks.

Transformation can be accomplished with either Agrobacterium or withparticle bombardment methods. An expression vector is created containinga constitutive plant promoter and the cDNA of the gene in a pUC vector.The plasmid DNA is prepared from E. coli cells using with Qiagen kitaccording to manufacturer's instruction. Approximately 2 g ofembryogenic callus is spread in the center of a sterile filter paper ina Petri dish. An aliquot of liquid MSO with 10 g/l sucrose is added tothe filter paper. Gold particles (1.0 μm in size) are coated withplasmid DNA according to method of Sanford et al., 1993 and aredelivered to the embryogenic callus with the following parameters: 500μg particles and 2 μg DNA per shot, 1300 psi and a target distance of8.5 cm from stopping plate to plate of callus and 1 shot per plate ofcallus.

After the bombardment, calli are transferred back to the fresh callusdevelopment medium and maintained in the dark at room temperature for a1-week period. The callus is then transferred to growth conditions inthe light at 25° C. to initiate embryo differentiation with theappropriate selection agent, e.g. 250 nM Arsenal, 5 mg/l PPT or 50 mg/LKanamycin. Shoots resistant to the selection agent are appearing andonce rooted are transferred to soil.

Samples of the primary transgenic plants (T0) are analyzed by PCR toconfirm the presence of T-DNA. These results are confirmed by Southernhybridization in which DNA is electrophoresed on a 1% agarose gel andtransferred to a positively charged nylon membrane (Roche Diagnostics).The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

Transgenic T0 ryegrass plants are propagated vegetatively by excisingtillers. The transplanted tillers are maintained in the greenhouse for 2months until well established. The shoots are defoliated and allowed togrow for 2 weeks.

Example 14 Engineering Soybean Plants by Over-Expressing YNL090W, e.g.from Saccharomyces cerevisiae or Plants

Soybean can be transformed according to the following modification ofthe method described in the Texas A&M patent U.S. Pat. No. 5,164,310.Several commercial soybean varieties are amenable to transformation bythis method. The cultivar Jack (available from the Illinois SeedFoundation) is commonly used for transformation. Seeds are sterilized byimmersion in 70% (v/v) ethanol for 6 min and in 25% commercial bleach(NaOCl) supplemented with 0.1% (v/v) Tween for 20 min, followed byrinsing 4 times with sterile double distilled water. Removing theradicle, hypocotyl and one cotyledon from each seedling propagatesseven-day seedlings. Then, the epicotyl with one cotyledon istransferred to fresh germination media in petri dishes and incubated at25° C. under a 16-hr photoperiod (approx. 100 μE-m-2s-1) for threeweeks. Axillary nodes (approx. 4 mm in length) are cut from 3-4 week-oldplants. Axillary nodes are excised and incubated in AgrobacteriumLBA4404 culture.

Many different binary vector systems have been described for planttransformation (e.g. An, G. in Agrobacterium Protocols. Methods inMolecular Biology vol 44, pp 47-62, Gartland K M A and M R Davey eds.Humana Press, Totowa, N.J.). Many are based on the vector pBIN19described by Bevan (Nucleic Acid Research. 1984. 12:8711-8721) thatincludes a plant gene expression cassette flanked by the left and rightborder sequences from the Ti plasmid of Agrobacterium tumefaciens. Aplant gene expression cassette consists of at least two genes—aselection marker gene and a plant promoter regulating the transcriptionof the cDNA or genomic DNA of the trait gene. Various selection markergenes can be used as described above, including the Arabidopsis geneencoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat.Nos. 57,673,666 and 6,225,105). Similarly, various promoters can be usedto regulate the trait gene to provide constitutive, developmental,tissue or environmental regulation of gene transcription as describedabove. In this example, the 34S promoter (GenBank Accession numbersM59930 and X16673) is used to provide constitutive expression of thetrait gene.

After the co-cultivation treatment, the explants are washed andtransferred to selection media supplemented with 500 mg/L timentin.Shoots are excised and placed on a shoot elongation medium. Shootslonger than 1 cm are placed on rooting medium for two to four weeksprior to transplanting to soil.

The primary transgenic plants (T0) are analyzed by PCR to confirm thepresence of T-DNA. These results are confirmed by Southern hybridizationin which DNA is electrophoresed on a 1% agarose gel and transferred to apositively charged nylon membrane (Roche Diagnostics). The PCR DIG ProbeSynthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and is used as recommended by themanufacturer.

Example 14c Engineering Corn Plants by Over-Expressing YNL090W, e.g.from Saccharomyces cerevisiae or Plants

Transformation of maize (Zea Mays L.) is performed with a modificationof the method described by Ishida et al. (1996. Nature Biotech14745-50). Transformation is genotype-dependent in corn and onlyspecific genotypes are amenable to transformation and regeneration. Theinbred line A188 (University of Minnesota) or hybrids with A188 as aparent are good sources of donor material for transformation (Fromm etal. 1990 Biotech 8:833-839), but other genotypes can be usedsuccessfully as well. Ears are harvested from corn plants atapproximately 11 days after pollination (DAP) when the length ofimmature embryos is about 1 to 1.2 mm. Immature embryos areco-cultivated with Agrobacterium tumefaciens that carry “super binary”vectors and transgenic plants are recovered through organogenesis. Thesuper binary vector system of Japan Tobacco is described in WO patentsWO94/00977 and WO95/06722. Vectors can be constructed as described.Various selection marker genes can be used including the maize geneencoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat.No. 6,025,541). Similarly, various promoters can be used to regulate thetrait gene to provide constitutive, developmental, tissue orenvironmental regulation of gene transcription. In this example, the 34Spromoter (GenBank Accession numbers M59930 and X16673) is used toprovide constitutive expression of the trait gene.

Excised embryos are grown on callus induction medium, then maizeregeneration medium, containing imidazolinone as a selection agent. ThePetri plates are incubated in the light at 25° C. for 2-3 weeks, oruntil shoots develop. The green shoots are transferred from each embryoto maize rooting medium and incubated at 25° C. for 2-3 weeks, untilroots develop. The rooted shoots are transplanted to soil in thegreenhouse. T1 seeds are produced from plants that exhibit tolerance tothe imidazolinone herbicides and which are PCR positive for thetransgenes.

The T1 generation of single locus insertions of the T-DNA can segregatefor the transgene in a 3:1 ratio. Those progeny containing one or twocopies of the transgene are tolerant of the imidazolinone herbicide.Homozygous T2 plants can exhibited similar phenotypes as the T1 plants.Hybrid plants (F1 progeny) of homozygous transgenic plants andnon-transgenic plants can also exhibited increased similar phenotyps.

Example 14d Engineering Wheat Plants by Over-Expressing YNL090W, e.g.from Saccharomyces cerevisiae or Plants

Transformation of wheat is performed with the method described by Ishidaet al. (1996 Nature Biotech. 14745-50). The cultivar Bobwhite (availablefrom CYMMIT, Mexico) is commonly used in transformation. Immatureembryos are co-cultivated with Agrobacterium tumefaciens that carry“super binary” vectors, and transgenic plants are recovered throughorganogenesis. The super binary vector system of Japan Tobacco isdescribed in WO patents WO94/00977 and WO95/06722. Vectors wereconstructed as described. Various selection marker genes can be usedincluding the maize gene encoding a mutated acetohydroxy acid synthase(AHAS) enzyme (U.S. Pat. No. 6025541). Similarly, various promoters canbe used to regulate the trait gene to provide constitutive,developmental, tissue or environmental regulation of gene transcription.In this example, the 34S promoter (GenBank Accession numbers M59930 andX16673) can be used to provide constitutive expression of the traitgene.

After incubation with Agrobacterium, the embryos are grown on callusinduction medium, then regeneration medium, containing imidazolinone asa selection agent. The Petri plates are incubated in the light at 25° C.for 2-3 weeks, or until shoots develop. The green shoots are transferredfrom each embryo to rooting medium and incubated at 25° C. for 2-3weeks, until roots develop. The rooted shoots are transplanted to soilin the greenhouse. T1 seeds are produced from plants that exhibittolerance to the imidazolinone herbicides and which are PCR positive forthe transgenes.

The T1 generation of single locus insertions of the T-DNA can segregatefor the transgene in a 3:1 ratio. Those progeny containing one or twocopies of the transgene are tolerant of the imidazolinone herbicide.Homozygous T2 plants exhibited similar phenotypes.

Example 14e Engineering Rapeseed/Canola Plants by Over-ExpressingYNL090W, e.g. from Saccharomyces cerevisiae or Plants

Cotyledonary petioles and hypocotyls of 5-6 day-old young seedlings areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can be used.

Agrobacterium tumefaciens LBA4404 containing a binary vector are usedfor canola transformation. Many different binary vector systems havebeen described for plant transformation (e.g. An, G. in AgrobacteriumProtocols. Methods in Molecular Biology vol 44, pp 47-62, Gartland K M Aand M R Davey eds. Humana Press, Totowa, N.J.). Many are based on thevector pBIN19 described by Bevan (Nucleic Acid Research. 1984.12:8711-8721) that includes a plant gene expression cassette flanked bythe left and right border sequences from the Ti plasmid of Agrobacteriumtumefaciens. A plant gene expression cassette consists of at least twogenes—a selection marker gene and a plant promoter regulating thetranscription of the cDNA or genomic DNA of the trait gene. Variousselection marker genes can be used including the Arabidopsis geneencoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat.Nos. 57,673,666 and 6,225,105). Similarly, various promoters can be usedto regulate the trait gene to provide constitutive, developmental,tissue or environmental regulation of gene transcription. In thisexample, the 34S promoter (GenBank Accession numbers M59930 and X16673)can be used to provide constitutive expression of the trait gene.

Canola seeds are surface-sterilized in 70% ethanol for 2 min., and thenin 30% Clorox with a drop of Tween-20 for 10 min, followed by threerinses with sterilized distilled water. Seeds are then germinated invitro 5 days on half strength MS medium without hormones, 1% sucrose,0.7% Phytagar at 23° C., 16 hr. light. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andare inoculated with Agrobacterium by dipping the cut end of the petioleexplant into the bacterial suspension. The explants are then culturedfor 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7%Phytagar at 23° C., 16 hr light. After two days of co-cultivation withAgrobacterium, the petiole explants are transferred to MSBAP-3 mediumcontaining 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l)for 7 days, and then cultured on MSBAP-3 medium with cefotaxime,carbenicillin, or timentin and selection agent until shoot regeneration.When the shoots are 5-10 mm in length, they are cut and transferred toshoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots ofabout 2 cm in length are transferred to the rooting medium (MS0) forroot induction.

Samples of the primary transgenic plants (T0) are analyzed by PCR toconfirm the presence of T-DNA. These results are confirmed by Southernhybridization in which DNA is electrophoresed on a 1% agarose gel andare transferred to a positively charged nylon membrane (RocheDiagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) isused to prepare a digoxigenin-labelled probe by PCR, and used asrecommended by the manufacturer.

Example 14f Engineering Alfalfa Plants by Over-Expressing YNL090W Genes,e.g. from Saccharomyces cerevisiae or E. coli or Plants

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659).

Petiole explants are cocultivated with an overnight culture ofAgrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 PlantPhysiol 119: 839-847) or LBA4404 containing a binary vector. Manydifferent binary vector systems have been described for planttransformation (e.g. An, G. in Agrobacterium Protocols. Methods inMolecular Biology vol 44, pp 47-62, Gartland K M A and M R Davey eds.Humana Press, Totowa, N.J.). Many are based on the vector pBIN19described by Bevan (Nucleic Acid Research. 1984. 12:8711-8721) thatincludes a plant gene expression cassette flanked by the left and rightborder sequences from the Ti plasmid of Agrobacterium tumefaciens. Aplant gene expression cassette consists of at least two genes—aselection marker gene and a plant promoter regulating the transcriptionof the cDNA or genomic DNA of the trait gene. Various selection markergenes can be used including the Arabidopsis gene encoding a mutatedacetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. Nos. 57,673,666 and6,225,105). Similarly, various promoters can be used to regulate thetrait gene that provides constitutive, developmental, tissue orenvironmental regulation of gene transcription. In this example, the 34Spromoter (GenBank Accession numbers M59930 and X16673) can be used toprovide constitutive expression of the trait gene.

The explants are cocultivated for 3 d in the dark on SH induction mediumcontaining 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings are transplantedinto pots and grown in a greenhouse.

The T0 transgenic plants are propagated by node cuttings and rooted inTurface growth medium. The plants are defoliated and grown to a heightof about 10 cm (approximately 2 weeks after defoliation).

Example 14g Engineering Alfalfa Plants by Over-Expressing YLR375W Genes,e.g. from Saccharomyces cerevisiae or Plants

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659).

Petiole explants are cocultivated with an overnight culture ofAgrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 PlantPhysiol 119: 839-847) or LBA4404 containing a binary vector. Manydifferent binary vector systems have been described for planttransformation (e.g. An, G. in Agrobacterium Protocols. Methods inMolecular Biology vol 44, pp 47-62, Gartland K M A and M R Davey eds.Humana Press, Totowa, N.J.). Many are based on the vector pBIN19described by Bevan (Nucleic Acid Research. 1984. 12:8711-8721) thatincludes a plant gene expression cassette flanked by the left and rightborder sequences from the Ti plasmid of Agrobacterium tumefaciens. Aplant gene expression cassette consists of at least two genes—aselection marker gene and a plant promoter regulating the transcriptionof the cDNA or genomic DNA of the trait gene. Various selection markergenes can be used including the Arabidopsis gene encoding a mutatedacetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. Nos. 57,673,666 and6,225,105). Similarly, various promoters can be used to regulate thetrait gene that provides constitutive, developmental, tissue orenvironmental regulation of gene transcription. In this example, the 34Spromoter (GenBank Accession numbers M59930 and X16673) can be used toprovide constitutive expression of the trait gene.

The explants are cocultivated for 3 d in the dark on SH induction mediumcontaining 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings are transplantedinto pots and grown in a greenhouse.

The T0 transgenic plants are propagated by node cuttings and rooted inTurface growth medium. The plants are defoliated and grown to a heightof about 10 cm (approximately 2 weeks after defoliation).

Equivalents

Those of ordinary skill in the art will recognize, or will be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the claims.

1. A process for the production of fine chemical, which comprises a)increasing or generating the biological activity represented by aprotein as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,382, 384, 386, 388, 390, 392 or 394 in a non-human organism, or in oneor more parts thereof; and b) growing the organism under conditionswhich permit the production of the fine chemical in said organism.
 2. Aprocess for the production of fine chemical comprising increasing orgenerating in an organism or a part thereof the expression of at leastone nucleic acid molecule comprising a nucleic acid molecule selectedfrom the group consisting of: a) nucleic acid molecule encoding of thepolypeptide as depicted in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392 or 394 or a fragment thereof, whichconfers an increase in the amount of fine chemical in an organism or apart thereof; b) nucleic acid molecule comprising of the nucleic acidmolecule as depicted in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295,297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351,353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379,381, 383, 385, 387, 389, 391 or 393; c) nucleic acid molecule whosesequence can be deduced from a polypeptide sequence encoded by a nucleicacid molecule of (a) or (b) as a result of the degeneracy of the geneticcode and conferring an increase in the amount of fine chemical in anorganism or a part thereof; d) nucleic acid molecule which encodes apolypeptide which has at least 50% identity with the amino acid sequenceof the polypeptide encoded by the nucleic acid molecule of (a) to (c)and conferring an increase in the amount of fine chemical in an organismor a part thereof; e) nucleic acid molecule which hybridizes with anucleic acid molecule of (a) to (c) under stringent hybridizationconditions and conferring an increase in the amount of fine chemical inan organism or a part thereof; f) nucleic acid molecule whichencompasses a nucleic acid molecule which is obtained by amplifyingnucleic acid molecules from a cDNA library or a genomic library usingthe primers in SEQ ID NO: 53 or SEQ ID NO: 54 and conferring an increasein the amount of the fine chemical in an organism or a part thereof; g)nucleic acid molecule encoding a polypeptide which is isolated with theaid of monoclonal antibodies against a polypeptide encoded by one of thenucleic acid molecules of (a) to (f) and conferring an increase in theamount of fine chemical in an organism or a part thereof; h) nucleicacid molecule encoding a polypeptide comprising the consensus sequenceas depicted in SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 397, SEQ ID NO: 398, SEQ IDNO: 399 and/or SEQ ID NO: 400 and conferring an increase in the amountof the fine chemical in an organism or a part thereof; and i) nucleicacid molecule which is obtainable by screening a suitable nucleic acidlibrary under stringent hybridization conditions with a probe comprisingone of the sequences of the nucleic acid molecule of (a) to (k) or witha fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule characterizedin (a) to (k) and conferring an increase in the amount of the finechemical in an organism or a part thereof, or a nucleotide sequencecomplementary thereto.
 3. The process of claim 2 further comprisingrecovering the free or bound fine chemical.
 4. The process of claim 2further comprising the following steps: a) selecting an organism or apart thereof expressing the polypeptide encoded by the nucleic acidmolecule characterized in claim 2; b) mutagenizing the selected organismor the part thereof; c) comparing the activity or the expression levelof said polypeptide in the mutated organism or the part thereof with theactivity or the expression of said polypeptide of the selected organismor the part thereof; d) selecting the mutated organism or the partthereof, which comprises an increased activity or expression level ofsaid polypeptide compared to the selected organism or the part thereof;e) optionally, growing and cultivating organism or the part thereof; andf) recovering, and optionally isolating, the free or bound fine chemicalproduced by the selected mutated organism or the part thereof.
 5. Theprocess of claim 2, wherein the activity of said protein or theexpression of said nucleic acid molecule is increased or generatedtransiently or stably.
 6. An isolated nucleic acid molecule comprising anucleic acid molecule selected from the group consisting of: a) nucleicacid molecule encoding of the polypeptide as depicted in SEQ ID NO: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340,342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392 or 394 or afragment thereof, which confers an increase in the amount of finechemical in an organism or a part thereof; b) nucleic acid moleculecomprising of the nucleic acid molecule as depicted in SEQ ID NO: 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285,287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313,315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341,343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391 or 393 or afragment thereof, which confers an increase in the amount of finechemical in an organism or a part thereof; c) nucleic acid moleculewhose sequence can be deduced from a polypeptide sequence encoded by anucleic acid molecule of (a) or (b) as a result of the degeneracy of thegenetic code and conferring an increase in the amount of fine chemicalin an organism or a part thereof; d) nucleic acid molecule which encodesa polypeptide which has at least 50% identity with the amino acidsequence of the polypeptide encoded by the nucleic acid molecule of (a)to (c) and conferring an increase in the amount of fine chemical in anorganism or a part thereof; e) nucleic acid molecule which hybridizeswith a nucleic acid molecule of (a) to (c) under stringent hybridizationconditions and conferring an increase in the amount of fine chemical inan organism or a part thereof; f) nucleic acid molecule whichencompasses a nucleic acid molecule which is obtained by amplifyingnucleic acid molecules from a cDNA library or a genomic library usingthe primers in SEQ ID NO: 53 or SEQ ID NO: 54 and conferring an increasein the amount of the fine chemical in an organism or a part thereof; g)nucleic acid molecule encoding a polypeptide which is isolated with theaid of monoclonal and/or polyclonal antibodies against a polypeptideencoded by one of the nucleic acid molecules of (a) to (f) andconferring an increase in the amount of fine chemical in an organism ora part thereof; h) nucleic acid molecule encoding a polypeptidecomprising the consensus sequence as depicted in SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399 and/or SEQ ID NO: 400 andconferring an increase in the amount of the fine chemical in an organismor a part thereof; and i) nucleic acid molecule which is obtainable byscreening a suitable nucleic acid library under stringent hybridizationconditions with a probe comprising one of the sequences of the nucleicacid molecule of (a) to (k) or with a fragment thereof having at least15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of thenucleic acid molecule characterized in (a) to (k) and conferring anincrease in the amount of the fine chemical in an organism or a partthereof, whereby the nucleic acid molecule distinguishes over thesequence as depicted in SEQ ID NO: 1, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327,329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355,357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,385, 387, 389, 391 or 393 by one or more nucleotides.
 7. A nucleic acidconstruct comprising one or more regulatory elements which confers theexpression of the nucleic acid molecule of claim
 6. 8. A vectorcomprising the nucleic acid molecule as claimed in claim
 6. 9. Thevector as claimed in claim 8, wherein the nucleic acid molecule is inoperable linkage with regulatory sequences for the expression in aprokaryotic or eukaryotic host.
 10. A host cell transformed stably ortransiently with the nucleic acid molecule as claimed in claim
 6. 11.The host cell of claim 10, wherein the host cell is a transgenic hostcell.
 12. The host cell of claim 10, which wherein the host cell is aplant cell, an animal cell, a microorganism, a yeast cell, a funguscell, a prokaryotic cell, an eukaryotic cell or an archaebacterium. 13.A process for producing a polypeptide, wherein the process comprisesstably or transiently transforming the nucleic acid molecule as claimedin claim 6 into a host cell, and expressing the polypeptide encoded bysaid nucleic acid molecule in the host cell.
 14. The polypeptideproduced by the process as claimed in claim 13 wherein the polypeptidedistinguishes over the sequence as depicted in SEQ ID NO: 2, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,378, 380, 382, 384, 386, 388, 390, 392 or 394 by one or more aminoacids.
 15. An antibody, which binds specifically to the polypeptideencoded by a nucleic acid sequence as claimed in claim 6a).
 16. A plant,or plant tissue, propagation material, or harvested material thereofcomprising the host cell as claimed in claim 12, wherein the host cellis a plant cell or an Agrobacterium.
 17. A method for screening foragonists and antagonists of the activity of a polypeptide encoded by thenucleic acid molecule of claim 6 conferring an increase in the amount offine chemical in an organism or a part thereof comprising: (a)contacting cells, tissues, plants or microorganisms which express apolypeptide encoded by the nucleic acid molecule of claim 6 conferringan increase in the amount of the fine chemical in an organism or a partthereof with a candidate compound or a sample comprising a plurality ofcompounds under conditions which permit the expression of thepolypeptide; (b) assaying the fine chemical level or the polypeptideexpression level in the cells, tissues, plants or microorganisms or amedia wherein the cells, tissues, plants or microorganisms are culturedor maintained; and (c) identifying the agonist or antagonist bycomparing the measured fine chemical level or polypeptide expressionlevel with a standard fine chemical or polypeptide expression levelmeasured in the absence of said candidate compound or said samplecomprising said plurality of compounds, wherein an increased level overthe standard indicates that the compound or the sample comprising saidplurality of compounds is an agonist and a decreased level over thestandard indicates that the compound or the sample comprising saidplurality of compounds is an antagonist.
 18. A process for theidentification of a compound conferring increased fine chemicalproduction in a plant or microorganism, comprising the steps: a)culturing a plant cell or tissue or microorganism or maintaining a plantexpressing the polypeptide encoded by the nucleic acid molecule of claim6 conferring an increase in the amount of the fine chemical in anorganism or a part thereof, and utilizing a readout system which iscapable of interacting with the polypeptide under suitable conditionswhich permit the interaction of the polypeptide with said readout systemin the presence of a compound or a sample comprising a plurality ofcompounds, and said readout system is capable of providing a detectablesignal in response to the binding of a compound to said polypeptideunder conditions which permit the expression of said readout system andthe expression of the polypeptide encoded by the nucleic acid moleculeof claim 6 conferring an increase in the amount of the fine chemical inan organism or a part thereof; b) identifying if the compound is aneffective agonist by detecting the presence or absence or increase of asignal produced by said readout system.
 19. A method for theidentification of a gene product conferring an increase in the finechemical production in a cell, comprising the following steps: a)contacting the nucleic acid molecules of a sample, which can contain acandidate gene encoding a gene product conferring an increase in finechemical after expression with the nucleic acid molecule of claim 6; b)identifying the nucleic acid molecules, which hybridize under relaxedstringent conditions with the nucleic acid molecule of claim 6; c)introducing the candidate nucleic acid molecules in host cellsappropriate for producing the fine chemical; d) expressing theidentified nucleic acid molecules in the host cells; e) assaying thefine chemical level in the host cells; and f) identifying nucleic acidmolecule and its gene product which expression confers an increase inthe fine chemical level in the host cell after expression compared tothe wild type.
 20. A method for the identification of a gene productconferring an increase in fine chemical production in a cell, comprisingthe following steps: a) identifying in a data bank nucleic acidmolecules of an organism; which can contain a candidate gene encoding agene product conferring an increase in the fine chemical amount or levelin an organism or a part thereof after expression, and which are atleast 30% homolog to the nucleic acid molecule of claim 6; b)introducing the candidate nucleic acid molecules in host cellsappropriate for producing the fine chemical; c) expressing theidentified nucleic acid molecules in the host cells; d) assaying thefine chemical level in the host cells; and e) identifying the nucleicacid molecule and its gene product which expression confers an increasein the fine chemical level in the host cell after expression compared tothe wild type.
 21. A method for the production of an agriculturalcomposition comprising identifying a compound according to the method ofclaim 17, and formulating the identified compound in a form acceptablefor an application in agriculture. 22-24. (canceled)
 25. Food or feedcomposition comprising the nucleic acid molecule of claim 6, a nucleicacid construct or a vector thereof, a polypeptide encoded thereby or anantibody which specifically binds to said polypeptide, a plant, planttissue or harvested material thereof, or a host cell containing saidnucleic acid molecule.
 26. (canceled)
 27. A composition comprising thenucleic acid molecule of claim 6, a polypeptide encoded thereby, or anantibody which specifically binds to said polypeptide, or a nucleic acidconstruct or a vector thereof, and optionally an agricultural acceptablecarrier.
 28. A composition comprising an antagonist or agonist of theactivity of a polypeptide encoded by the nucleic acid molecule of claim6 conferring an increase in the amount of fine chemical in an organismor a part thereof.
 29. Food or feed composition comprising theantagonist or agonist identified according to claim
 17. 30. A method foridentifying plant varieties having increased capacity for production ofa fine chemical, wherein the method comprises using the nucleic acidmolecules as claimed in claim 6 in mapping and/or breeding processes,and identifying plant varieties which have increased production of saidfine chemical.